Immunogenic Substances Comprising A Polyinosinic Acid-Polycytidilic Acid Based Adjuvant

ABSTRACT

The present invention provides a polynucleotide adjuvant (PICKCa) composition and methods of use in eliciting an immune response, in particular a mucosal immune response. The polynucleotide adjuvant comprises of a polyriboinosinic-polyribocytidylic acid (PIC), at least one antibiotic and at least one positive ion. The present invention also provides an immunogenic composition comprising the polynucleotide adjuvant composition together with other immunogenic compositions such as an antigen (e.g., as in a vaccine) selected from viral, bacterial, fungal, parasitic and/or cancer antigens. The present invention further contemplates methods of use of such adjuvant compositions, particularly in eliciting an immune response, in particular a mucosal immune response to an antigenic compound.

FIELD OF INVENTION

The invention generally relates to immunogenic compositions and methodsof their use. More specifically the invention relates to an immunogeniccomposition comprising a polynucleotide adjuvant in combination with oneor more antigenic substances to be used to elicit disease specificimmune response in a host.

BACKGROUND OF INVENTION

The immune system may exhibit both specific and nonspecific immunity.Nonspecific immunity encompasses various cells and mechanisms such asphagocytosis (the engulfing of foreign particles or antigens) bymacrophages or granulocytes, and natural killer (NK) cell activity,among others. Nonspecific immunity relies on mechanisms lessevolutionarily advanced and does not display the acquired nature ofspecificity and memory, which are exemplary hallmarks of a specificimmune response. The key differences between specific and nonspecificimmunity are based upon B and T cell specificity. These cellspredominantly acquire their responsiveness after activation with aspecific antigen and have mechanisms to display memory in the event offuture exposure to that specific antigen. As a result, vaccination(involving specificity and memory) is an effective protocol to protectagainst harmful pathogens.

Generally, B and T lymphocytes, which display specific receptors ontheir cell surface for a given antigen, produce specific immunity. Thespecific immune system may respond to different antigens in two ways: 1)humoral-mediated immunity, which includes B cell stimulation andproduction of antibodies or immunoglobulins, antigen and helper T cells(predominantly Th2), and 2) cell-mediated immunity, which generallyinvolves T cells including cytotoxic T lymphocytes (CTLs), althoughother cells are also involved in the generation of a CTL response (e.g.,antigen presenting cells and Th1 cells).

In the continual pursuit for safer and more effective vaccines, newtechnologies, including recombinant, purification and synthetic methods,have been used to improve the quality and specificity of antigens used.Purified, sub-unit and synthesized antigens demonstrate increased safetybut diminished immunogenicity which has been one driver for theidentification of effective adjuvant. Thus an effective adjuvant isincreasingly an essential component of modern vaccines. Adjuvants aregenerally compounds, that when administered with an antigen (either inconjunction with, or given prior to the administration of the antigen)enhances and/or modifies the immune response to that particular antigen.

Exemplary adjuvants that have been used to enhance an immune responseinclude aluminum compounds (all generally referred to as “Alum”),oil-in-water emulsions (complete Freund's adjuvant (CFA) is anoil-in-water emulsion containing dried, heat-killed Mycobacteriumtuberculosis organisms), Saponin (isolated from the bark of QuillajaSaponoria, the adjuvant active component known as Quile A), CpG ODN(synthetic oligodeoxynucleotide containing unmethylated CpGdinucleotides), monophosphoryl lipid A (MPL) derived from thelipopolysaccharide of Salmonella Minnesota Re595, Liposomes (usuallymade up of biodegradable materials such as phospholipids) andbiodegradable polymer microspheres (made from a variety of polymers suchas, polyphosphazene and polyanhydrides). The adjuvant properties ofthese compounds have been evaluated with each adjuvant showingadvantages and disadvantages.

Polynucleotide complexes have been investigated for their variousapplications including acting as adjuvants. Double-stranded RNAs(dsRNAs) are very potent biologic modifiers that can exert a profoundinfluence on cells at nanomolar concentrations. The modulating effectsof dsRNA include a broad spectrum of actions at the molecular andcellular levels.

At the molecular level, dsRNAs can elicit biological effects such asinterferon synthesis, induction of protein kinase, enhancement ofhistocompatibility antigen and inhibition of metabolism. And at thecellular level, dsRNA can elicit biological effects such aspyrogenicity, mitogenicity, macrophage activation, activation of humoralimmunity, activation of cell-mediated immunity and induction ofantiviral state. Immunomodulating effects of dsRNAs has been disclosed.U.S. Pat. No. 4,124,702 disclosed that double stranded polynucleotidesinduced interferon induction in living animal cells. U.S. Pat. No.3,906,092 disclosed that the antibody response to an adjuvant typevaccine was augmented by incorporation in the vaccine of apolynucleotide or a complex of polynucleotides. Houston et al.established PICLC (polyinosinic acid polycytidylic acidpoly-L-lysinecarboxy-methylcellulose complex) as a potent adjuvant byincreasing primary antibody response without the aid of an additionaladjuvant.

Polyinosinic acid-polycytidylic acid (PIC), one of most studiedpolynucleotide complexes, was not effective when used in monkeys andhumans due to its instability in the body after administration. Thus,PIC has been modified in many ways to overcome one or anotherdeficiency. For example, a complex of polyriboinosinic-polyribocytidylicacid with poly-L-lysine hydrobromide is about 5 to 15 times as resistantto hydrolysis by pancreatic ribonuclease as the parent PIC.

Lin et al. described that an antiviral drug comprising polyinosinicpolycytidylic acid, kanamycin and calcium can be used as an adjuvant(Lin, et al., A new immunostimulatory complex (PICKCa) in experimentalrabies: antiviral and adjuvant effects, Arch Virol, 131: 307-19, 1993;and Chinese Patent No. 93105862.7). The Chinese Patent No. 93105862.7provides for the use of the general composition of Poly I:C, kanamycinand calcium (PICKCa) as an adjuvant in a vaccine for human and mammalianapplication. However, Lin found that that the form of PICKCa originallyidentified does not provide the optimal efficacy/safety profile for useas an adjuvant and also induces unacceptable adverse side effects undercertain conditions.

The present invention provides novel immunogenic compositions thatexhibit improved safety and efficacy profiles; and methods of use ofsuch compositions. Subject immunogenic compositions include apolynucleotide adjuvant and an antigen.

LITERATURE

The following references may be of interest:

-   JP 1093540A2;-   U.S. Pat. No. 4,124,702-   U.S. Pat. No. 3,692,899-   U.S. Pat. No. 3,906,092-   U.S. Pat. No. 4,389,395-   U.S. Pat. No. 4,349,538-   U.S. Pat. No. 4,024,241-   U.S. Pat. No. 3,952,097-   Houston et al., Infection and Immunity, 14: 318-9, 1976C-   Wright and Adler-Moore, Biochemical and Biophysical Research    Communications, 131: 949-45, 1985-   Lin, et al., A new immunostimulatory complex (PICKCa) in    experimental rabies: antiviral and adjuvant effects, Arch Virol,    131: 307-19, 1993-   Chinese Patent 93105862.7-   Gupta R. K. et al., Adjuvants—a balance between toxicity and    adjuvanticity, Vaccine, 11:293-306, 1993-   Arnon, R. (Ed.) Synthetic Vaccines 1:83-92, CRC Press, Inc., Boca    Raton, Fla., 1987-   Sela, M., Science 166:1365-1374 (1969)-   U.S. Pat. No. 6,008,200-   Ellouz et al., Biochem. & Biophy. Res. Comm., 59:1317, 1974-   U.S. Pat. No. 4,094,971-   U.S. Pat. No. 4,101,536-   U.S. Pat. No. 4,153,684-   U.S. Pat. No. 4,235,771-   U.S. Pat. No. 4,323,559-   U.S. Pat. No. 4,327,085-   U.S. Pat. No. 4,185,089-   U.S. Pat. No. 4,082,736-   U.S. Pat. No. 4,369,178-   U.S. Pat. No. 4,314,998-   U.S. Pat. No. 4,082,735-   U.S. Pat. No. 4,186,194-   U.S. Pat. No. 6,468,558-   New Trends and Developments in Vaccines, edited by Voller et al.,    University Park Press, Baltimore, Md., USA, 1978-   Klein, J., et al., Immunology (2nd), Blackwell Science Inc., Boston    (1997)-   Gupa R. K. and Siber G. R., Adjuvants for human vaccines—current    status, problems and future prospects, Vaccine, 13 (14): 1263-1276,    1995-   Richard T Kenney et al. Meeting Report—2^(nd) meeting on novel    adjuvants currently in/close to human clinical testing, Vaccine 20    2155-2163, 2002-   Laboratory Techniques in Rabies Edited by F X Meslin, M M Kaplan, H    Koprowski 4^(th), 1996, Edition ISBN 92 4 1544 1

SUMMARY OF THE INVENTION

In general, the present invention relates to novel immunogeniccompositions comprising a polynucleotide adjuvant composition togetherwith an immunogenic or antigenic substance, and methods of use ineliciting an immune response.

Accordingly, there is provided an immunogenic composition comprising:(a) a polynucleotide adjuvant comprising: apolyriboinosinic-polyribocytidylic acid (PIC), at least one anantibiotic, and at least one positive ion; and (b) at least oneantigen;wherein the composition is formulated for sustained releaseadministration.

The immunogenic composition according to the invention may comprise apolynucleotide adjuvant composition molecules heterogeneous formolecular weight, wherein the molecular weight is at least 66,000Daltons.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—ELISPOT detection of murine splenocytes producing interferon-γafter immunization with vaccines comprising PIKA and/or HBsAg adw

FIG. 2—ELISPOT detection of murine splenocytes producing IL-2 afterimmunization with vaccines comprising PIKA and/or HBsAg adw

FIG. 3—ELISPOT detection of IL-4 produced by murine splenocytes afterimmunization with vaccines comprising PIKA and/or HBsAg adw

FIG. 4—ELISA detection of specific IgG titers from murine serum (diluted400×) after immunization with vaccines comprising PIKA and/or HBsAg adw

FIG. 5—ELISPOT detection of murine splenocytes producinginterferon-gamma (γ) after immunization with vaccines comprising PIKAand/or inactivated split influenza antigen

FIG. 6—ELISPOT detection of murine splenocytes producing IL-2 afterimmunization with vaccines comprising PIKA and/or inactivated splitinfluenza antigen

FIG. 7—ELISPOT detection of murine splenocytes producing IL-4 afterimmunization with vaccines comprising PIKA and/or inactivated splitinfluenza antigen

FIG. 8—ELISA detection of specific IgG titers from murine serum (diluted900×) after immunization with vaccines comprising PIKA and/orinactivated split influenza antigen

FIG. 9—ELISPOT detection of murine splenocytes producing interferon-γafter immunization with vaccines comprising PIKA and/or HIV gp120antigen

FIG. 10—ELISPOT detection of murine splenocytes producing IL-2 afterimmunization with vaccines comprising PIKA and/or HIV gp120 antigen

FIG. 11—ELISPOT detection of murine splenocytes producing IL-4 afterimmunization with vaccines comprising PIKA and/or HIV gp120 antigen

FIG. 12—FACS analysis of murine splenocytes after immunization withvaccines comprising PIKA and/or HIV gp120 antigen, percentage of CD4+vecells expressing interferon-γ

FIG. 13—ELISPOT detection of murine splenocytes producing interferon-γafter immunization with vaccines comprising PIKA and/or anthrax rPAantigen

FIG. 14—ELISPOT detection of murine splenocytes producing IL-2 afterimmunization with vaccines comprising PIKA and/or anthrax rPA antigen

FIG. 15—ELISPOT detection of murine splenocytes producing IL-4 afterimmunization with vaccines comprising PIKA and/or anthrax rPA antigen

FIG. 16—FACS analysis of murine splenocytes after immunization withvaccines comprising PIKA and/or anthrax rPA antigen, percentage ofCD4+ve cells expressing interferon-γ

FIG. 17—ELISA detection of specific IgG titers from murine serum(diluted 400×) after immunization with vaccines comprising PIKA and/oranthrax rPA antigen

FIG. 18—ELISA detection of specific IgG titers from murine serum 16weeks after immunization with vaccines comprising PIKA and/or anthraxrPA antigen

FIG. 19—ELISPOT detection of murine splenocytes producing interferon-γafter immunization with vaccines comprising PIKA and/or HSV 2gD antigen

FIG. 20—ELISPOT detection of murine splenocytes producing IL-2 afterimmunization with vaccines comprising PIKA and/or HSV 2gD antigen

FIG. 21—ELISPOT detection of murine splenocytes producing IL-4 afterimmunization with vaccines comprising PIKA and/or HSV 2gD antigen

FIG. 22—FACS analysis of murine splenocytes after immunization withvaccines comprising PIKA and/or HSV 2gD antigen, percentage of CD4+vecells expressing interferon-γ

FIG. 23—ELISA detection of specific IgG titers from murine serum(diluted 2,700×) after immunization with vaccines comprising PIKA and/orHSV 2 gD antigen

FIG. 24—ELISPOT detection of murine splenocytes producing interferon-γafter immunization with vaccines comprising PIKA and/or inactivated H5N1antigen

FIG. 25—ELISPOT detection of murine splenocytes producing IL-2 afterimmunization with vaccines comprising PIKA and/or inactivated H5N1antigen

FIG. 26—ELISPOT detection of murine splenocytes producing IL-4 afterimmunization with vaccines comprising PIKA and/or inactivated H5N1antigen

FIG. 27—FACS analysis of murine splenocytes after immunization withvaccines comprising PIKA and/or inactivated H5N1 antigen, percentage ofCD4+ve cells expressing interferon-γ

FIG. 28—ELISA detection of specific IgG titers from murine serum(diluted 900×) after immunization with vaccines comprising PIKA and/orinactivated H5N1 antigen

FIG. 29—ELISA detection of specific IgG titers from murine serum(diluted 16,000) after immunization with vaccines comprising PIKA and/orwhole inactivated SARS antigen

FIG. 30—ELISA detection of specific antibody H5 titers from chickenserum after immunization with vaccines comprising PIKA and/orinactivated H5N1 antigen

FIG. 31—ELISA detection of specific H9 antibody titers from chickenserum after immunization with vaccines comprising PIKA and/orinactivated H5N1 antigen

FIG. 32—Survival rate of mice exposed to wild rabies virus andsubsequent treatment with rabies vaccine

FIG. 33—ELISA detection of specific antibody titers from murine serumafter immunization with vaccines comprising PIKA and/or HBsAg adw

FIG. 34—ELISA detection of specific antibody titers from murine serumafter immunization with vaccines comprising PIKA and/or inactivatedsplit influenza antigen

FIG. 35—ELISA detection of specific IgG1 titers from murine serum afterimmunization with vaccines comprising PIKA and/or HBsAg

FIG. 36—ELISA detection of specific IgG2a titers from murine serum afterimmunization with vaccines comprising PIKA and/or HBsAg

FIG. 37—ELISPOT detection of murine splenocytes producing interferon-γafter immunization with vaccines comprising PIKA and/or HBsAg

FIG. 38—ELISPOT detection of murine splenocytes (post restimulation 6days with 2 ug/ml IPQ peptide) producing interferon-γ after immunizationwith vaccines comprising PIKA and/or HBsAg

Table 1 provides a table of exemplary viral pathogens which can serve asa source of antigen and diseases associated with these organisms.

Table 2 provides a table of exemplary bacterial pathogens which canserve as a source of antigen and diseases associated with theseorganisms.

Table 3 provides a table of exemplary fungal pathogens which can serveas a source of antigen and diseases associated with these organisms.

Table 4 provides a table of exemplary parasites which can serve as asource of antigen and diseases associated with these organisms.

Table 5 provides a table of exemplary cancers (e.g., by tissue type)which serve as a source of antigen.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of certain embodiments of the inventionand the Examples included herein.

Throughout this application, where publications are referenced, thedisclosures of these publications are hereby incorporated by reference,in their entireties, into this application in order to describe morefully the state of art to which this invention pertains.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skilledin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “animmunogenic composition” includes a plurality of such compositions andreference to “the antigen” includes reference to one or more antigensand equivalents thereof known to those skilled in the art, and so forth.It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

DEFINITIONS OF TERMS

Prior to setting forth details of the present invention it may be usefulto an understanding thereof to set forth definitions of several termsthat are used herein.

The term “adjuvant,” as used herein, refers to any substance or mixtureof substances that increases or diversifies the immune response of ahost to an antigenic compound. Specifically:

-   -   1. The term “PICKCa” generally refers to a composition of poly        I:C, kanamycin and calcium irrespective of particular physical        and immunogenic properties.    -   2. “Av-PICKCa” refers to a form of PICKCa used commercially as        an antiviral drug.    -   3. “PIKA” refers to a composition of the invention comprising        poly I:C, an antibiotic (e.g., kanamycin), and a positive ion        (e.g., calcium), where the PIKA is characterized by physical        characteristics (e.g., molecular weight, size, and the like)        such that upon administration, PIKA exhibits characteristics of        an adjuvant with reduced adverse side effects (e.g., reduced        toxicity) relative to, for example, PICKCa and greater potency        (e.g., stimulates an enhanced immune response) relative to, for        example, Av-PICKCa.

The term “Poly I:C” or “PIC” refers to a composition comprisingpolyriboinosinic and polyribocytidylic nucleic acids, which may also bereferred to as polyinosinic acid-polycytidylic acid, respectively.

“PIC-containing molecule” or “PIC-containing compound” refers to,without limitation, PIC, which may be optionally complexed or otherwisecombined with at least one or both of an antibiotic (e.g., kanamycin)and a positive ion (e.g., calcium) present in a composition comprisingthe PIC-containing molecule. In one embodiment, the PIC-containingmolecule does not include poly-L-lysine or a derivative thereof in thecomplex.

“Heterogeneous” as used herein in the context of the adjuvantcompositions of the invention indicates that components of thecomposition, e.g., the PIC-containing molecules, are not uniform withrespect to a physical characteristic of molecular weight, size, or both.Where a composition is described as heterogenous for a given physicalcharacteristic, and is further described by a range of values for thatphysical characteristic, the composition is said to be composedsubstantially of molecules characterized by molecules having a physicalcharacteristic that is distributed within and across the recited range.While the composition may not contain a molecule representative of everyphysical characteristic value within the upper and lower limits of arecited range, the composition will generally include at least onemolecule having the physical characteristic of the upper value and ofthe lower value. The composition in certain embodiments may includemolecules outside the stated range of physical characteristics used todescribe the composition. The molecules that are present in thecomposition outside the prescribed range do not materially affect thebasic and novel characteristics of the composition.

The term “individual,” used interchangeably herein with “host,”“subject,” and “animal,” includes humans and all domestic, e.g.livestock and pets, and wild mammals and fowl, including, withoutlimitation, cattle, horses, cows, swine, sheep, goats, dogs, cats,rabbits, deer, mink, chickens, ducks, geese, turkeys, game hens, and thelike.

The term “antibody” includes polyclonal and monoclonal antibodies, aswell as antigenic compound binding fragments of such antibodiesincluding Fab, F(ab′)2, Fd, Fv fragments, and single chain derivativesof the same. In addition, the term “antibody” includes naturallyoccurring antibodies as well as non-naturally occurring antibodies,including, for example, chimeric, bifunctional and humanized antibodies,and related synthetic isoforms. The term “antibody” is usedinterchangeably with “immunoglobulin.”

As used herein, the term “antigenic compound” refers to any substancethat can be recognized by the immune system (e.g., bound by an antibodyor processed so as to elicit a cellular immune response) underappropriate conditions.

An “antigen” as used herein includes but is not limited to cells; cellextracts; proteins; lipoproteins; glycoproteins; nucleoproteins;polypeptides; peptides; polysaccharides; polysaccharide conjugates;peptide mimics of polysaccharides; lipids; glycolipids; carbohydrates;viruses; viral extracts; bacteria; bacterial extracts; fungi; fungalextracts; multicellular organisms such as parasites; and allergens.Antigens may be exogenous (e.g., from a source other than the individualto whom the antigen is administered, e.g., from a different species) orendogenous (e.g., originating from within the host, e.g., a diseasedelement of body, a cancer antigen, a virus infected cell producingantigen, and the like). Antigens may be native (e.g.,naturally-occurring); synthetic; or recombinant. Antigens include crudeextracts; whole cells; and purified antigens, where “purified” indicatesthat the antigen is in a form that is enriched relative to theenvironment in which the antigen normally occurs and/or relative to thecrude extract, for example, a cultured form of the antigen.

An “immunogenic composition” as used here in refers to a combination oftwo or more substances (e.g., an antigen and an adjuvant) that togetherelicit an immune response when administered to a host.

The term “polypeptide,” “peptide,” “oligopeptide,” and “protein,” areused interchangeably herein, and refer to a polymeric form of aminoacids of any length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones.

An “effective amount of an antigenic compound” refers to an amount ofantigenic compound which, in optional combination with an adjuvant, willcause the subject to produce a specific immunological response to theantigenic compound.

The term “immune response” refers to any response to an antigenic orimmunogenic compound by the immune system of a vertebrate subject.Exemplary immune responses include, but are not limited to local andsystemic cellular as well as humoral immunity, such as cytotoxic Tlymphocytes (CTL) responses, including antigen-specific induction ofCD8⁺ CTLs, helper T-cell responses including T-cell proliferativeresponses and cytokine release, and B-cell responses including antibodyresponse.

The term “eliciting an immune response” is used herein generally toencompass induction and/or potentiation of an immune response.

The term “inducing an immune response” refers to an immune response thatis stimulated, initiated, or induced.

The term “potentiating an immune response” refers to a pre-existingimmune response that is improved, furthered, supplemented, amplified,enhanced, increased or prolonged.

The expression “enhanced immune response” or similar means that theimmune response is elevated, improved or enhanced to the benefit of thehost relative to the prior immune response status, for example, beforethe administration of an immunogenic composition of the invention.

The terms “humoral immunity” and “humoral immune response” refer to theform of immunity in which antibody molecules are produced in response toantigenic stimulation.

The terms “cell-mediated immunity” and “cell-mediated immune response”are meant to refer to the immunological defense provided by lymphocytes,such as that defense provided by T cell lymphocytes when they come intoclose proximity to their victim cells. A cell-mediated immune responsenormally includes lymphocyte proliferation. When “lymphocyteproliferation” is measured, the ability of lymphocytes to proliferate inresponse to a specific antigen is measured. Lymphocyte proliferation ismeant to refer to B cell, T-helper cell or cytotoxic T-lymphocyte (CTL)cell proliferation.

The term “immunogenic amount” refers to an amount of antigenic compoundsufficient to stimulate an immune response, when administered with asubject immunogenic composition, as compared with the immune responseelicited by the antigen in the absence of the polynucleotide adjuvant.

The term “immunopotentiating amount” refers to the amount of theadjuvant needed to effect an increase in antibody titer and/orcell-mediated immunity when administered with an antigenic compound in acomposition of the invention, as compared with the increase in antibodyand/or cell mediated immunity level observed in the absence of thepolynucleotide adjuvant.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in asubject, particularly a mammalian subject, more particularly a human,and includes: (a) preventing the disease or symptom from occurring in asubject which may be predisposed to the disease or symptom but has notyet been diagnosed as having it; (b) inhibiting the disease symptom,e.g., arresting its development; or relieving the disease symptom, i.e.,causing regression of the disease or symptom (c) reduction of a level ofa product produced by the infectious agent of a disease (e.g., a toxin,an antigen, and the like); and (d) reducing an undesired physiologicalresponse to the infectious agent of a disease (e.g., fever, tissueedema, and the like).

As used herein, the term “mixing” includes any method to combine thecomponents of the composition; such methods include, but are not limitedto, blending, dispensing, dissolving, emulsifying, coagulating,suspending, or otherwise physically combining the components of thecomposition.

A “pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; or (2) salts formed whenan acidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine, andthe like.

The term “unit dosage form” as used herein refers to physically discreteunits suitable as unitary dosages for human and animal subjects, eachunit containing a predetermined quantity of compounds of the presentinvention calculated in an amount sufficient to produce the desiredeffect in association with a pharmaceutically/physiologically acceptablediluent, carrier or vehicle.

Exemplary Embodiments of the Invention

The present invention is directed to immunogenic compositions andmethods useful for the induction and/or enhancement of an immuneresponse, which may be humoral and/or cell-mediated, in a human, anon-human animal, or cell culture. In general, a subject immunogeniccomposition comprises an antigen (an “antigenic composition”) and anadjuvant. The presence of the adjuvant enhances or modifies the immuneresponse to the antigen. The adjuvant may alter the quality of theimmune response by affecting the subclasses (isotypes) ofimmunoglobulins, chemokines, and/or cytokines produced. As a result theinnate immunity, humoral and/or cell-mediated immune responses are moreeffective with the presence of the adjuvant.

A particular advantage is the effectiveness of the PIKA adjuvant incombination with an antigenic substance in inducing a specific humoralimmune response thereby enhancing protective immunity.

A further important advantage is that the PIKA adjuvant in combinationwith an antigen can induce a specific cell mediated immune response thatis essential for a therapeutic vaccine for limiting and treatingintracellular viral, bacterial and parasite infections as well as forchronic diseases therapies such as the treatment of cancers orauto-immune disease.

Accordingly, included in the invention are compositions having theunique product attributes that make them most suitable for use asvaccines to be administered to animals and/or humans that address theneed for a safe adjuvant, which elicits a beneficial immune response.

Accordingly, the present invention provides an adjuvant and immunogeniccompositions that can be used safely in humans and animals.

Accordingly, there is provided an immunogenic composition comprising:(a) a polynucleotide adjuvant comprising: apolyriboinosinic-polyribocytidylic acid (PIC), at least one anantibiotic, and at least one positive ion; and (b) at least oneantigen;wherein the composition is formulated for sustained releaseadministration.

The immunogenic composition according to the invention may comprise apolynucleotide adjuvant composition molecules heterogeneous formolecular weight, wherein the molecular weight is at least 66,000Daltons. The value of 66,000 Daltons corresponds to the size of about6.4 Svedbergs. Accordingly, a molecular weight range of 66,000 to1,200,000 Daltons corresponds to the size from about 6.4 to 24.0Svedbergs.

In some embodiments, a PIKA adjuvant composition comprising apolynucleotide, an antibiotic and a positive ion, wherein thepolynucleotide may be polyriboinosinic-polyribocytidylic acid (PIC); theantibiotic may be kanamycin, and the ion may be calcium.

In one aspect of particular interest, the invention provides for animmunogenic composition for enhancing the antigenicity of an antigeniccompound comprising the polynucleotide adjuvant composition that iscapable of eliciting an antigen specific cell mediated immune response.

In one aspect of particular interest, the invention provides for animmunogenic composition for enhancing the antigenicity of an antigeniccompound comprising the polynucleotide adjuvant composition that iscapable of eliciting an antigen specific humoral immune response.

In one aspect of particular interest, the invention provides for animmunogenic composition for enhancing the antigenicity of an antigeniccompound comprising the polynucleotide adjuvant composition that iscapable of eliciting a combined specific cell mediated and humoralimmune response.

In one aspect of particular interest, the invention provides for anadjuvant composition or immunogenic composition comprising an adjuvantcomposition wherein the adjuvant composition or the immunogeniccomposition is freeze-dried.

In one aspect of particular interest, the invention provides for the useof a polynucleotide adjuvant composition for the preparation of amedicament for enhancing the immunogenic response of a host.

Polynucleotide Adjuvant

A subject immunogenic composition comprises a PIC-containingpolynucleotide adjuvant, e.g., a PIKA composition, is generally composedof polyinosinic acid, polycytidylic acid, an antibiotic (e.g.,kanamycin), and a divalent cation (e.g., calcium). It will be understoodthat reference to PIKA herein is exemplary of such PIC-containingadjuvants.

PIC-containing adjuvants of interest can be manufactured using methodsavailable in the art. The PIC-containing adjuvant composition can bemanufactured through any appropriate process. For example thepolynucleotide adjuvant composition can be manufactured by mixing ofpolyinosinic acid, polycytidylic acid, an antibiotic and the source of apositive ion in a sodium chloride/phosphate buffer solution that has apH between pH6 and pH8. The polyinosinic acid and polycytidylic acid aregenerally provided at a concentration of 0.1 to 10 mg/ml, usually 0.5 to5 mg/ml and more usually 0.5 to 2.5 mg/ml. The hyperchromicity valueshould be greater than 10%, greater than 15%, greater than 20%, orgreater than 50%. The preparation of the PIC and the combination withthe antibiotic (e.g., kanamycin) and the positive ion (e.g., calcium) isgenerally conducted under quality standards consistent withinternational Good Manufacturing Process.

In certain embodiments of the present invention, the antibioticcomponent of the adjuvant is kanamycin. Where the antibiotic iskanamycin, in some embodiments, the kanamycin in the polynucleotideadjuvant composition is used together with or substituted by one or moreantibiotics selected from the group including tobramycin,anthracyclines, butirosin sulfate, gentamicins, hygromycin, amikacin,dibekacin, nebramycin, metrzamide, neomycin, puromycin, streptomycin andstreptozocin. The antibiotic (e.g., Kanamycin or the like) in thepolynucleotide adjuvant composition of the invention is generallyprovided at a concentration of from about 10 units/ml to 100,000units/ml, from about 100 units/ml to 10,000 units/ml, or from about 500units/ml to 5,000 units/ml.

In certain embodiments of the present invention, the polynucleotideadjuvant composition further comprises a positive ion (cation), usuallya divalent cation, normally a cation of an alkali metal. The positiveion is generally provided in the composition of the invention as asource of positive ions such as a salt or complex, e.g., an organic orinorganic salt or complex, usually an inorganic salt or organic complex.Exemplary positive ions include, but are not necessarily limited to,calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt,deuterium, gallium, iodine, iron, or zinc.

The positive ion can be provided in the form of any suitable salt ororganic complex, including, but not necessarily limited to chloride,fluoride, hydroxide, phosphate, or sulfate salts. For example, where thepositive ion is calcium, the ion can be in the form of calciumcarbonate, calcium chloride, calcium fluoride, calcium hydroxide,calcium phosphates, or calcium sulfate.

The positive ion (e.g. calcium) can be provided in the composition ofthe invention at a concentration in the range of from about 10 umol to10 mmol/ml, usually from about 50 umol to 5 mmol/ml, and more usuallyfrom about 100 umol to 1 mmol/ml. The term “μmol” is used throughout torefer to micromole.

Where the positive ion in the adjuvant composition of the invention iscalcium, it can be in combination with or substituted by other positiveions, including cadmium, lithium, magnesium, cerium, cesium, chromium,cobalt, deuterium, gallium, iodine, iron, and zinc, wherein the ions canbe in the form of inorganic salts or organic complexes.

The resulting composition is a PIC-containing adjuvant that furthercontains an antibiotic and a positive ion. In a particular embodiment,where the antibiotic is kanamycin and the ion is calcium the product maybe described as PICKCa. In a related embodiment the PICKCa compositionmay contain molecules without restriction of different physicalcharacteristics.

PIKA Adjuvant Composition

In a particular exemplary embodiments, the PIC-containing adjuvant isPIKA. PIKA may be produced in a variety of ways, with production fromPICKCa being of particular interest. PIKA can be produced from PICKCathrough additional manufacturing processes that involve the isolationand/or concentration of molecules of a defined molecular size and/orweight. The separation and concentration of polynucleotide molecules ofparticular characteristics using filtration, chromatography, thermaltreatment, centrifugal separation, electrophoresis, and similar methodsthat are standard processes and are known to those skilled in the art.

In embodiments of particular interest, the invention features anadjuvant generally referred to as PIKA comprising apolyriboinosinic-polyribocytidylic acid (PIC), an antibiotic (e.g.,kanamycin), and a positively charged ion (e.g., a calcium ion), whereinthe composition contains molecules of the adjuvant heterogeneous formolecular weight having a molecular weight of from about 66,000 to1,200,000 Daltons. That is, the adjuvant composition comprises moleculeswith a weight distribution in the range of from about 66,000 to1,200,000 Daltons.

In related embodiments, the PIKA polynucleotide adjuvant compositionmolecules in the composition are heterogeneous, that is the weight ofthe adjuvant molecules are distributed within a range of molecularweight, where the molecular weight is from about 300,000 to 1,200,000Daltons, or from about 66,000 to 660,000 Daltons, or from about 300,000to 660,000 Daltons, or from about 300,000 to 2,000,000 Daltons, or fromabout 66,000 Daltons to about 100,000 Daltons, 100,000 to 200,000Daltons, from about 300,000 Daltons to about 4,000,000 Daltons, or fromabout 500,000 Daltons to 1,000,000 Daltons, or from about 1,000,000Daltons to 1,500,000 Daltons, or from about 1,500,000 Daltons to2,000,000 Daltons, or from about 2,000,000 Daltons to 2,500,000 Daltons,or from about 2,500,000 Daltons to 3,000,000 Daltons, or from about3,000,000 Daltons to 3,500,000 Daltons, or from about 3,500,000 Daltonsto 4,000,000 Daltons, or from about 4,000,000 Daltons to 4,500,000Daltons, or from about 4,500,000 Daltons to 5,000,000 Daltons.

In related embodiments, the PIKA polynucleotide adjuvant compositionmolecules in the composition have an average molecular weight equal orequal to or greater than 66,000 Daltons, greater than 150,000 Daltons,or equal to or greater than 250,000 Daltons, or equal to or greater than350,000 Daltons, or equal to or greater than 500,000 Daltons, or equalto or greater than 650,000 Daltons, or equal to or greater than 750,000Daltons, or equal to or greater than 1,000,000 Daltons, or equal to orgreater than 1,200,000 Daltons, or equal to or greater than 1,500,000Daltons, or equal to or greater than 2,000,000 Daltons.

In embodiments of particular interest, the invention features anadjuvant generally referred to as PIKA comprising apolyriboinosinic-polyribocytidylic acid (PIC), an antibiotic, and apositive ion wherein the composition contains molecules of the adjuvantheterogeneous, that is the size of the adjuvant molecules aredistributed within a range of molecular size, for molecular size havinga sediment co-efficient Svedbergs (S) of from about 6.43 S to 24.03 S.

In related embodiments, the PIKA polynucleotide adjuvant compositionmolecules in the composition are heterogeneous, that is the size of theadjuvant molecules are distributed within a range of molecular size,where the molecular size is from about 12.8 S to 24.03 S, or from about3 S to 12 S or from about 6.43 to 18.31 S, or from about 12.8 to 18.31S, or from about 12.8 S to 30.31 S, or from about 12.8 S to 41.54 S, orfrom about 13.5 S, to 18.31 S, or from about 13.5 S to 24.03 S, or fromabout 16.14 to 22.12 S, or from about 22.12 S to 26.6 S, or from about26.6 S to 30.31 S, or from about 30.31 S to 33.55 S, or from about 33.55S to 36.45 S, or from about 36.45 S to 39.1 S, or from about 39.1 S to41.54 S, or from about 41.54 S to 43.83 S, or from about 43.83 S to45.95 S.

In further related embodiments, the PIKA polynucleotide adjuvantcomposition has an average sedimentation co-efficient (Svedbergs)greater than 9, or greater than 12, or greater than 13.5, or greaterthan 15, or greater than 16, or greater than 17, or greater than 18, orgreater than 19, or greater than 20, or greater than 21, or greater than22 or greater than 25, or greater than 30.

Immunogenic Properties

An immunogenic composition, including PIKA and an antigen, can generallyinduce an antigen-specific immune response in at least two ways: i)humoral-mediated immunity, which includes B cell stimulation andproduction of antibodies or immunoglobulins (other cells are alsoinvolved in the generation of an antibody response, e.g.antigen-presenting cells, including macrophages and helper T cells (Th1and Th2), and ii) cell-mediated immunity, which generally involves Tcells including cytotoxic T lymphocytes, although other cells are alsoinvolved in the generation of a cytotoxic T lymphocyte response (e.g.,Th1 and/or Th2 cells and antigen presenting cells).

Furthermore, the polynucleotide adjuvant composition may alter thequality of the immune response by affecting the subclasses (isotypes) ofimmunoglobulins produced, as well as their affinities.

The degree and nature of the immunogenic response induced by a subjectimmunogenic composition may be thus assessed by measuring the presenceof molecules including cytokines, chemokines and antibodies produced bycells of the immune system.

Interleukin-4 is mainly produced by activated Th2 cells. The productionof interleukin-4 (IL-4) induces the activation of B cells and therebythe production of IgG1 and IgE immunoglobulins (antibodies) which may bemeasured in the samples of blood serum. IL-4 is considered as anindictor and typical cytokine of Th2 immune response. Th2 cells tend topromote antibody response.

Interleukin-2 (IL-2) is mainly produced by activated Th1 cell as well asNK and lymphokine-activated killer (LAK) cells. IL-2 is instrumental inthe proliferation and maturing of T cells an essential stage in aneffective cell mediated adaptive immune response.

Interferon-γ (INF-γ), which may be produced by a variety of cellsincluding natural killer cells as well as both CD4⁺ and CD8⁺ T cells,plays an essential part in the adaptive immune response including theactivation of macrophages to become highly microbicidal. Further, INF-γis an influencing factor in directing the development of specificallyTh1 T cells thereby up-regulating a cell mediated adaptive immuneresponse.

The invention contemplates methods of use of the polynucleotide adjuvantof the invention with an antigen, for example, to elicit an antigenspecific humoral response and/or specific cellular (e.g., T cell)response in a subject. The immune response elicited may be a response toan antigen in a naïve subject, or may serve to enhance an existingimmune response (e.g., as in a booster). It has been found thatimmunogenic compositions according to the invention comprising PIKA haveparticularly advantageous properties as described herein.

A variety of different antigens were tested in vivo for their ability toinduce an immune response with and without the PIKA adjuvant. Theantigens tested include: a recombinant protein hepatitis B surfaceantigen type adw, an inactivated split influenza vaccine (VAXIGRIP fromSanofi Pasteur), a synthesized HIV peptide antigen, a recombinantprotein herpes simplex virus type 2 gD antigen, recombinant protectiveanthrax protein antigen, inactivated whole virus avian influenza antigenstrain H5N1 and an inactivated whole virus Severe Acute RespiratorySyndrome (SARS) inactivated antigen.

In each case presence of the PIKA adjuvant together with the antigenenhanced the expression of cytokines when compared with the antigen orPIKA alone. In particular the enhanced expressions of the cytokinesINF-γ, IL-2 and IL-4 (see Examples 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6)indicates the stimulation of a specific adaptive immunity was greaterwith the presence of the PIKA adjuvant and more specifically theenhanced expressions of the cytokines INF-γ, IL-2 indicates thepredominant Th1 cell immunity was significantly improved with thepresence of the PIKA adjuvant. The activity of a cell mediated immuneresponse is a key feature essential for treating intracellular viral,bacterial and parasite infection and particularly important factor fordeveloping a therapeutic vaccine.

Further the composition containing PIKA stimulated INF-γ production byCD4+ T cells (Examples 1.3, 1.4, 1.5 and 1.6). This feature validatesthat PIKA is enhancing the adaptive immune response of the host.

The observed proliferation of antibodies in the blood serum demonstratesthat the PIKA adjuvant induces a beneficial humoral response. Increasedspecific antibody IgG titers were observed with the addition of PIKA toan immunogenic composition (see Examples 1.1, 1.2, 1.4, 1.5, 1.6, 2, 3,5 and 6).

The PIKA adjuvant enhances the immune response in a host when combinedwith a inactivated antigen (Examples 1.2, 1.6, 2, 3, 4 and 6), a peptideantigen (Example 1.3) and a recombinant antigen (Examples 1.1, 1.4, 1.5,5 and 7).

A particular feature of the PIKA adjuvant is to provide adequateprotection to both limit and/or eradicate infection in a host, and/or toreduce the risk of symptoms of a disease that could result frominfection by a pathogen. VAXIGRIP (Sanofi Pasteur) used as an antigen inExamples 1.2 and 6 is itself a human influenza vaccine that elicits adegree of immune activity considered sufficient to provide protectionagainst an actual influenza infection. The addition of PIKA to VAXIGRIPfurther enhanced the immune response as demonstrated by the degree ofbeneficial cytokines (IL-2, INF-γ and IL-4) and specific IgG expressedby the immune system.

In a further demonstration of PIKA's protective properties, 24 ten-dayold chickens were inoculated with a composition comprising PIKA andinactivated avian influenza antigens including strains H5N1 and H9N2(Example 3). The chickens were subsequently challenged with the liveH5N1 virus and observed for a two week period. At the end of the programthe survival rate for the chickens inoculated with the PIKA/antigencomposition was 83% as compared with only 17% for a control group of 24chickens that were exposed to the live virus without prior inoculationwith the PIKA/antigen composition.

In a related experiment to demonstrate the therapeutic enhancingproperties of the PIKA adjuvant, Balb/c mice were challenged with astrain of wild rabies virus (Example 4). Post infection the threedifferent groups of animals were inoculated with a regime of treatmentswith different rabies vaccines. The survival rate of the group of miceinoculated with a combination of an inactivated purified hamster kidneycell rabies antigen plus PIKA attained 80%. The survival rate of thesecond group of mice administered with a hamster kidney cell purifiedrabies antigen with an alum adjuvant was 15%. Further the third group ofmice administered with the Sanofi-Aventis' “Verorab”—vero cellinactivated rabies vaccine had a survival rate of 20%.

In a further demonstration of the properties of the PIKA adjuvant,Example 7 demonstrates that the presence of PIKA in conjunction withHBsAg type adw enhances the production of specific IgG1 (Table 26 FIG.35) and more significantly IgG2a (Table 27 FIG. 36) titers in murineserum. The conclusion from this observation is that PIKA enhances atherapeutic immune response in particular a Th1 biased immune response.

In a related experiment (Example 7) the presence of PIKA in vaccineformulations comprising HBsAG type adw was shown to enhance theproduction of interferon-γ by splenocytes stimulated with a CD8 T cellpeptide epitope. This result demonstrates that PIKA induces a CD8+ Tcell immune response (Table 28 FIG. 37).

Further, (Example 7) the presence of PIKA in vaccine formulationscomprising HBsAg was shown to enhance the production of interferon-γ bysplenocytes cultured ex-vivo for six days with 2 ug/ml of the CD8 T cellpeptide epitope. This result demonstrates that PIKA induces a centralmemory T cell response.

Additional Features

In a further embodiment, a subject immunogenic composition is furtherdefined by the relative presence of the PIKA adjuvant and the antigen orantigens where the presence is measured in terms of one or morecharacteristics of quantity, concentration, volume, number of moleculesor other recognized metric.

In related embodiments, a subject immunogenic composition comprises apolynucleotide adjuvant composition and an antigen or antigens where thepresence of the adjuvant and the antigen in terms of weight or number ofmolecules is in a ratio of less than 1 to 1,000, of less than 1 to 900,of less than 1 to 800, of less than 1 to 700, of less than 1 to 500, ofless than 1 to 400, of less than 1 to 300, of less than 1 to 200, ofless than 1 to 100, of less than 1 to 50, of less than 1 to 10, of lessthan 1 to 5, of less than 1 to 2, of about 1 to 1, of greater than 2 to1, of greater than 5 to 1, of greater than 10 to 1, of greater than 50to 1, of greater than 100 to 1, of greater than 200 to 1, of greaterthan 300 to 1, of greater than 400 to 1, of greater than 500 to 1, ofgreater than 600 to 1, of greater than 700 to 1, of greater than 800 to1, of greater than 900 to 1, of greater than 1,000 to 1

In a further related embodiment a subject immunogenic composition isdefined in terms of dose; that is the quantity of vaccine that is to beadministered to induce the optimal beneficial immune response oralternatively the range of dose that may be administered from theminimum required to elicit an immune response to the maximum dose beyondwhich the incremental beneficial response is not medically justified inthe context of the potential inducement of adverse side effects.

In certain embodiments of particular interest the immunogeniccomposition comprises the polynucleotide adjuvant composition andantigen where the presence of the antigen in a unit dose is provided ina quantity, that is more than 0.1 ug, is more than 0.5 ug, is more than0.001 mg is more than 0.005 mg, is more than 0.01 mg, is more than 0.025mg, is more than 0.05 mg, is more than 0.075 mg, 0.1 mg is more than0.25 mg, is more than 0.5 mg, is more than 1.2 mg, is more than 1.4 mg,is more than 1.6 mg, is more than 1.8 mg, is more than 2.0 mg is morethan 2.5 mg, is more than 3 mg, is more than 3.5 mg, is more than 4 mg,is more than 5 mg, is more than 6 mg, is more than 7 mg, is more than 8mg, is more than 9 mg, is more than 10 mg, is more than 15 mg, is morethan 20 mg, is more than 25 mg, or is more than 50 mg.

An optimal amount of antigen and the optimal ratio of antigen to PIKAadjuvant can be ascertained by standard studies involving observationsof antibody titers and other immunogenic responses in the host.

Antigens

In an embodiment of particular interest the invention provides for apolynucleotide adjuvant composition together with an antigen or vaccinewhere the source of the antigen is a human antigen, a animal antigen, aplant antigen, one or more agents from infectious agents from any virus,bacteria including mycobacterium, fungus or parasite, cancer antigen,allergenic agents and other antigens, such as for developing autoimmunediseases.

In certain embodiments, the antigens may be derived from a naturalsource either crude or purified and used in its original live form orafter having been killed, or inactivated, or truncated, or attenuated,or transformed into a nonreverting form, or detoxified, or mutated intoa nontoxic form, or filtered or purified.

In some embodiments, the antigen is an isolated micro-organism antigenfor example, a viral antigen, a bacterial antigen, a fungal antigen, anallergy antigen, a cancer antigen or an autoimmune antigen. In otherembodiments, the antigen is a whole, inactivated antigen. Methods ofinactivating a whole antigens are well known in the art; any knownmethod can be used to inactivate an antigen and can be selectedappropriately for the type of antigen of interest. Such methods ofinactivating an antigen include for example, use of photoreactivecompounds; oxidizing agents; irradiation (e.g., UV irradiation;γ-irradiation); combinations of riboflavin and UV irradiation;solvent-detergent treatment (e.g., treatment with organic solventtri-N-butyl-phosphate with a detergent such as Tween 80); polyethyleneglycol treatment; pasteurization (heat treatment); and low pH treatment;mild enzymatic treatment with pepsin or trypsin; Methylene blue (MB)phototreatment; treatment with Dimethylmethylene blue (DMMB) and visiblelight; treatment with S-59, a psoralen derivative and UVA illumination;and the like.

In a related embodiment of particular interest the antigen may besynthesized by means of solid phase synthesis, or may be obtained bymeans of recombinant genetics, or may be otherwise manufacturedartificially so as to imitate the immunogenic properties of a pathogen.

Polypeptide antigens may be isolated from natural sources using standardmethods of protein purification known in the art, including, but notlimited to, liquid chromatography (e.g., high performance liquidchromatography, fast protein liquid chromatography, etc.), sizeexclusion chromatography, gel electrophoresis (including one-dimensionalgel electrophoresis, two-dimensional gel electrophoresis), affinitychromatography, or other purification technique. One may employ solidphase peptide synthesis techniques, where such techniques are known tothose of skill in the art. See Jones, The Chemical Synthesis of Peptides(Clarendon Press, Oxford)(1994). Generally, in such methods a peptide isproduced through the sequential additional of activated monomeric unitsto a solid phase bound growing peptide chain. Well-establishedrecombinant DNA techniques can be employed for production ofpolypeptides, such methods include, but are not limited to, for example,an expression construct comprising a nucleotide sequence encoding apolypeptide is introduced into an appropriate host cell (e.g., aeukaryotic host cell grown as a unicellular entity in in vitro cellculture, e.g., a yeast cell, an insect cell, a mammalian cell, etc.) ora prokaryotic cell (e.g., grown in in vitro cell culture), generating agenetically modified host cell; under appropriate culture conditions,the protein is produced by the genetically modified host cell.

In some embodiments, the antigen is a purified antigen, e.g., from about25% to 50% pure, from about 50% to about 75% pure, from about 75% toabout 85% pure, from about 85% to about 90% pure, from about 90% toabout 95% pure, from about 95% to about 98% pure, from about 98% toabout 99% pure, or greater than 99% pure.

The antigen may be acellular, capsular, infectious clone, replicon,vectored, microencapsulated, monovalent, bivalent or multivalent.

The polynucleotide adjuvant composition of the present invention canalso be utilized to enhance the immune response against antigensproduced by the use of DNA vaccines and/or DNA expressed proteins. TheDNA sequences in these vaccines coding for the antigen can be either“naked” or contained in a delivery system, such as liposomes.

In one aspect of particular interest a subject immunogenic compositionmay be defined by the selection of antigen or antigens that are used incombination with the PIKA adjuvant.

More specifically, the present invention provides for an immunogeniccomposition and method of use where the immunogenic compositioncomprises a PIKA adjuvant together with a viral antigen, whereinexemplary antigens include but are not limited to antigens of one ormore of the viruses described in Table 1.

TABLE 1 Viral pathogens and diseases Viral Taxonomy Disease 1Adenoviridae 2   Mastadenovirus 3    Human adenovirus A to F Common cold4 Arenaviridae 5   Old world arenaviruses 6    Ippy virus 7     Lassavirus Lassa fever 8     Lymphocytic choriomeningitis virus Lymphocyticchoriomeningitis disease 9 Astroviridae 10   Mamastrovirus 11     Humanastrovirus Gastroenteritis 12 Bunyaviridae 13  Hantavirus 14    Hantaanvirus Korean hemorrhagic fever 15   Nairovirus 16     Crimean-Congohemorrhagic fever virus Hemorrhagic Fevers 17   Orthobunyavirus 18    California encephalitis virus La Crosse Encephalitis 19  Phlebovirus 20     Rift Valley fever virus Rift Valley Fever 21Caliciviridae 22   Norovirus 23     Norwalk virus Diarrhea 24Flaviviridae 25   Flavivirus 26     Dengue virus group Dengue 27    Japanese encephalitis virus group 28      Japanese encephalitisvirus Japanese encephilitis 29       St. Louis encephalitis virus St.Louis encephalitis disease 30       West Nile virus West Nile virusdisease 31     tick-borne encephalitis virus group Tick-borneencephalitis 32     Yellow fever virus group Yellow fever 33  Hepacivirus 34     Hepatitis C virus Hepatitis C 35     Hepatitis Gvirus Hepatitis G 36  Hepadnaviridae 37     Orthohepadnavirus 38      Hepatitis B virus Hepatitis B 39 Hepatitis delta virus Hepatitis D40 Hepeviridae 41  Hepevirus 42       Hepatitis E virus Hepatitis E 43Herpesviridae 44   Alphaherpesvirinae 45    Simplexvirus 46      Cercopithecine herpesvirus 1 B Virus Infection 47       Humanherpesvirus 1 Herpes simplex type 1 48       Human herpesvirus 2 Herpessimplex type 2 49     Varicellovirus 50       Human herpesvirus 3(Varicella zoster virus) Chicken pox, Shingels 51   Betaherpesvirinae 52    Cytomegalovirus 53       Human herpesvirus 5 Cytomegalovirus (CMV)54     Roseolovirus 55      Human herpesvirus 6 Herpes 56       Humanherpesvirus 7 Herpes 57   Gammaherpesvirinae 58     Lymphocryptovirus 59      Human herpesvirus 4 Epstein-Barr virus Infection 60    Rhadinovirus 61       Human herpesvirus 8 Herpes 62 Mononegavirales63   Filoviridae 64     Ebola-like viruses 65       Ebola virus Eboladisease 66     Marburgvirus Marburg hemorrhagic fever 67 Paramyxoviridae 68    Paramyxovirinae 69       Henipavirus 70       Hendra virus Hendra virus disease 71       Morbillivirus 72        Measles virus Measles 73       Respirovirus 74         Humanparainfluenza virus 1 Human parainfluenza virus 75         Humanparainfluenza virus 3 Human parainfluenza virus 76       Rubulavirus 77        Human parainfluenza virus 2 Human parainfluenza virus 78        Human parainfluenza virus 4 Human parainfluenza virus 79        Mumps virus Mumps 80    Pneumovirinae 81      Metapneumovirus 82        Human metapneumovirus Human metapneumovirus 83       Pneumovirus84         Human respiratory syncytial virus Human respiratory syncytialdisease 85  Rhabdoviridae 86    Lyssavirus 87      Rabies virus Rabies88 Nidovirales 89  Coronaviridae 90    Coronavirus 91      Group 2species 92        Human coronavirus Coronovirus 93        SARScoronavirus SARS 94    Torovirus 95        Human torovirus Torovirusdisease 96 Picornaviridae 97  Aphthovirus 98     Equine rhinitis A virus99       Foot-and-mouth disease virus Foot-and-mouth disease virus 100  Enterovirus 101     Human enterovirus A 102         Humancoxsackievirus Human coxsackievirus 103         Human enterovirus Humanenterovirus 104     Human enterovirus B 105         Enterovirus Humanenterovirus 106         Human coxsackievirus Human coxsackievirus 107        Human echovirus Human echovirus 108     Human enterovirus C 109        Human coxsackievirus Human coxsackievirus 110     Humanenterovirus D 111         Human enterovirus Human enterovirus 112    Poliovirus 113         Human poliovirus Polio 114     Humanenterovirus sp. Human enterovirus 115     unclassified Enteroviruses 116        Human enterovirus sp. Human enterovirus 117   Hepatovirus 118    Hepatitis A virus Hepatitis A virus 119   Parechovirus 120     Humanparechovirus Human parechovirus 121         Human parechovirus 122  Rhinovirus (common cold viruses) 123     Human rhinovirus A 124        Human rhinovirus Common cold 125     Human rhinovirus B 126        Human rhinovirus Common cold 127     unclassified Rhinovirus 128        Human rhinovirus Common cold 129 Orthomyxoviridae 130   Influenzavirus A 131      Influenza A virus Influenza 132   Influenzavirus B 133      Influenza B virus Influenza 134   Influenzavirus C 135      Influenza C virus Influenza 136 Paramyxoviridae 137      Paramyxovirinae 138       Henipavirus 139        Hendra virus Hendra virus 140 Papillomaviridae 141   Alphapapillomavirus 142      Human papillomavirus Humanpapillomavirus 143   Betapapillomavirus 144     Human papillomavirusHuman papillomavirus 145   Gammapapillomavirus 146     Humanpapillomavirus Human papillomavirus 147   Mupapillomavirus 148     Humanpapillomavirus Human papillomavirus 149   unclassified Papillomaviridae150     Human papillomavirus types Human papillomavirus 151 Parvoviridae152   Parvovirinae 153     Erythrovirus 154       Human parvovirus 155      unclassified Erythrovirus 156         Human erythrovirus Humanerythrovirus 157 Polyomaviridae 158   Polyomavirus 159     JCpolyomavirus Progressive multifocal leukencephalopathy 160 Poxviridae161   Chordopoxvirinae 162     Orthopoxvirus 163       Monkeypox virusMonkey pox 164       Variola virus Smallpox 165 Reoviridae 166  Rotavirus 167     Rotavirus A Diarrhea 168     Rotavirus B Diarrhea169     Rotavirus C Diarrhea 170 Retroviridae 171   Orthoretrovirinae172     Deltaretrovirus 173       Primate T-lymphotropic virus 1 174        Human T-lymphotropic virus 1 Human T-lymphotropic virus 175      Primate T-lymphotropic virus 2 176         Human T-lymphotropicvirus 2 Human T-lymphotropic virus 177       Primate T-lymphotropicvirus 3 178         Human T-lymphotropic virus 3 Human T-lymphotropicvirus 179     Lentivirus 180       Primate lentivirus group 181        Human immunodeficiency virus type 1 and type 2 HIV 182  unclassified Retroviridae 183     Aids-associated retrovirus 184    Human endogenous retroviruses 185 Togaviridae 186   Alphavirus 187    EEEV complex 188      Eastern equine encephalitis virus Easternequine encephalitis 189    SFV complex 190      Chikungunya virusChikungunya fever 191     VEEV complex 192       Venezuelan equineencephalitis virus Venezuelan equine encephalitis 193     WEEV complex194       Western equine encephalomyelitis virus Western equineencephaalitis 195   Rubivirus 196     Rubella virus Rubella, GermanMeasels

More specifically, the present invention provides for an immunogeniccomposition and method of use where the immunogenic compositioncomprises a PIKA adjuvant together with a bacterial antigen, whereinexemplary antigens include but are not limited to antigens of one ormore of the bacteria described in Table 2.

TABLE 2 Bacterial pathogens and diseases Bacterial Taxonomy Disease 1Actinobacteria 2  Actinobacteria (class) (high G + C Gram-positivebacteria) 3   Acidimicrobidae 4    Actinobacteridae 5    Actinomycetales 6      Corynebacterineae 7       Corynebacteriaceae8        Corynebacterium 9         Corynebacterium diptheriae Diphtheria10   Actinobacteridae 11    Actinomycetales 12     Corynebacterineae 13     Mycobacteriaceae 14       Mycobacterium 15        Mycobacteriumabscessus Mycobacterium abscessus infection 16         Mycobacteriumavium complex Mycobacterium abscessus infection 17         Mycobacteriumleprae Leprosy/Hansen's Disease 18         Mycobacterium tuberculosisMycobacterium tuberculosis Infection 19      Nocardiadeae 20      Nocardia 21        Nocardia asteroides Nocardiosis 22       Nocardia farcinica Nocardiosis 23        Nocardia novaNocardiosis 24        Nocardia transvalensis Nocardiosis 25       Nocardia brasiliensis Nocardiosis 26        Nocardiapseudobrasiliensis Nocardiosis 27 28 Chlamydiae/Verrucomicrobia group 29 Chlamydiae 30   Chlamydiae (class) 31    Chlamydiales 32    Chlamydiaceae 33      Chlamydia 34       Chlamydia trachomatisChlamydia 35         Chlamydia pneumoniae Pneumonia 36         Chlamydiapsittaci Psittacosis 37         Chlamydia trachomatis, serovars A,Trachoma         B, Ba, and C 38         Chlamydophila pneumoniaePneumonia 39 Firmicutes (Gram-positive bacteria) 40  Bacilli 41  Bacillales 42    Bacillaceae 43     Bacillus 44      Bacillus cereusgroup 45       Bacillus anthracis Anthrax 46    Listeriaceae 47    Listeria 48      Listeria monocvtogenes Listeriosis 49   Staphylococcaceae 50     Staphylococcus 51      Staphylococcus aureusToxic Shock Syndrome 52      Staphylococcus aureus Methicillin ResistantStaphylococcus aureus (MRSA) 53      Staphylococcus aureus VISA and VRSAStaphylococcus aureus (VISA/VRSA) Infections 54   Lactobacillales 55   Streptococcaceae 56     Streptococcus Streptococcal Diseases 57     Group A streptococcus Scarlet Fever 58      Group B streptococcusMeningitis 59      Streptococcus pneumoniae Pneumonia 60  Clostridia 61  Clostridiales 62    Clostridaceae 63     Clostridium 64     Clostridium botulinum Botulism 65       Clostridium difficileDiarrhea 66       Clostridium tetani Tetanus Disease 67  Mollicutes 68  Mycoplasmatales 69    Mycoplasmataceae 70     Mycoplasma 71     Mycoplasma pneumonia Mycoplasma pneumoniae Infection 72Proteobacteria (purple bacteria and relatives) 73  Alphaproteobacteria74   Rhizobiales (rhizobacteria) 75    Bartonellaceae 76     Bartonella77      Bartonella henselae Cat Scratch Disease 78    Brucellaceae 79    Brucella Brucellosis 80 81    Rickettsiales (rickettsias) 82    Anaplasmataceae 83      Anaplasma Typhus Fevers 84      EhrlichiaTyphus Fevers 85     Rickettsiaceae 86      Rickettsieae 87      Orientia 88        Orientia tsutsugamushi Typhus Fevers 89      Rickettsia 90        Spotted fever group 91         Rickettsiarickettsii Rickettsia rickettsii Infection 92         Rickettsiaprowazekii Typhus Fevers 93         Rickettsia typhi Typhus Fevers 94 95 Betaproteobacteria 96   Burkholderiales 97    Alcaligenaceae 98    Bordetella 99      Bordetella pertussis Pertussis 100   Burkholderiaceae 101     Burkholderia 102      Burkholderia cepaciacomplex 103       Burkholderia cepacia Burkholderia cepacia Infection104       Burkholderia pseudomallei Melioidosis 105   Neisseriales 106   Neisseriaceae 107     Neisseria 108      Neisseria gonorrhoeaeGonorrhea 109       Neisseria meningitidis, meningococcus Meningitis 110 delta/epsilon subdivisions 111   Epsilonproteobacteria 112   Camplobacterales 113     Campylobacteraceae 114      CampylobacterCampylobacter Infection 115       Campylobacter jejuni Diarrhea 116    Helicobacteraceae 117      Heliobacter 118       Heliobacter pyloriHelicobacter pylori Infection 119  Gammaproteobacteria 120  Enterobacteriales 121    Entrobacteriaceae 122     Escherichia 123     Escherichia coli Dysentery 124     Salmonella Salmonellosis 125     Salmonella typhi Salmonella typhi Infection/Typhoid 126    Shigella 127      Shigella dysenteriae Dysentery 128      Shigellaflexneri Diarrhea 129      Shigella sonnei Shigellosis 130     YersiniaYersiniosis 131      Yersinia pestis Plague 132   Legionellales 133   Coxiellaceae 134     Coxiella 135      Coxiella burnetii Q Fever 136   Legionellaceae 137     Legionella 138      Legionella pneumophilaLegionellosis/Legionnaire's Disease 139      Legionella pneumophilaPontiac Fever 140   Pasteurellales 141    Pasteurellaceae 142    Haemophilus 143      Haemophilus ducreyi Haemophilus ducreyiInfection 144       Haemophilus influenzae serotype b Haemophilusinfluenzae Serotype b (Hib) Infection 145   Pseudomonadales 146   Pseudomonadaceae 147     Pseudomonas 148      Pseudomonas aeruginosagroup 149       Pseudomonas aeruginosa Pseudomonas aeruginosa infection150    Moraxellaceae 151     Acinobacter Acinetobacter Infection 152  Thiotrichales 153    Francisellaceae 154     Francisella 155     Francisella tularenis Francisella tularensis Infection 156  Vibrionales 157    Vibrionaceae 158     Vibrio 159      Vibrioparahaemolyticus Vibrio parahaemolyticus Infection 160      Vibriovulnificus Vibrio vulnificus Infection 161      Vibrio cholerae Cholera162 Spirochaetes 163  Spirochaetes (class) 164   Spirochaetales 165   Leptospiraceae 166     Leptospira Leptospirosis 167   Spirochaetaceae 168     Borrelia 169      Borrelia burgdorferi Group170       Borrelia burgdorferi Lyme Disease 171     Treponema 172     Treponema pallidun Syphilis

More specifically, the present invention provides for an immunogeniccomposition and method of use where the immunogenic compositioncomprises a PIKA adjuvant together with a fungal antigen, whereinexemplary antigens include but are not limited to antigens of one ormore of the fungi described in Table 3.

TABLE 3 Fungal pathogens and diseases Fungal Taxonomy Disease 1Ascomycota (ascomycetes) 2  Pezizomycotina 3   Eurotiomycetes 4   Eurotiales 5     Trichocomaceae 6      mitosporic Trichocomaceae 7      Aspergillus Aspergillosis 8    Onygenales 9     Ajellomycetaceae10      Ajellomyces 11       Ajellomyces capsulatus 12       Histoplasma capsulatum Histoplasmosis 13       Blastomycoidesdermatitidis Blastomycosis 14     Arthodermataceae Ringworm 15    mitosporic Onygenales 16      Coccidiodes 17       Coccidiodesimmitis Coccidioidomycosis, Valley fever 18     Paracoccidioides 19     Paracoccidioides brasiliensis 20   Sordariomycetes 21   Sordariomycetidae 22     Ophiostomatales 23      Ophiostomataceae 24      mitosporic Ophiostomataceae 25        Sporothrix schenckiiSporotrichosis 26  Pneumocystidomycetes 27   Pneumocystidales 28   Pneumocystidaceae 29     Pneumocystis 30      Pneumocystis jiroveciPCP Infection 31  Saccharomycotina 32   Saccharomycetes 33   Saccharomycetales 34     mitosporic Saccharomycetales 35      Candida36       Candida albicans Candidiasis, Thrush 37 Basidiomycota(basidiomycetes) 38  Hymenomycetes 39   Heterobasidiomycetes 40   Tremellomycetidae 41     Tremellales 42      Tremellaceae 43      Filobasidiella 44        Filobasidiella neoformans 45        Cryptococcus neoformans Cryptococcosis 46 Metoza 47  Eumetazoa48   Bilareria 49    Acoelomata 50     Platyhelminthes 51      Trematoda52       Digenae 53        Strigeidida 54         Schistosomatidea 55         Schistosoma 56           Schistosoma haematobiumSchistosomiasis 57           Schistosoma japonicum Schistosomiasis 58          Schistosoma mansoni Schistosomiasis 59    Pseudocoelomata 60    Nematoda 61      Chromadorae 62       Spirurida 63       Filarioidea 64         Onchocercidae 65          Brugia 66          Brugia malayi Lymohatic filariasis

More specifically, the present invention provides for an immunogeniccomposition and method of use, where the immunogenic compositioncomprises a PIKA adjuvant together with a parasitic antigen, whereinexemplary antigens include but are not limited to antigens of one ormore of the parasites described in Table 4.

TABLE 4 Parasitic pathogens and diseases Parasite Taxonomy DiseasePhylum Sarcomastigophora (the protozoa)  Subphylum Mastigophora (theflagellates)    Class Zoomastigophorea      Order Trichomonadida           Dientamoeba fragilis Dientamoeba fragilis Infection     Order Diplomonadida         Giardia lamblia (giardiasis) Giardiaintestinalis Giardiasis/Giardia Infection      Order Kinetoplastida           Leishmania Kala-Azar            Trypanosoma cruzi ChagasDisease            Trypanosoma brucei African Trypanosomiasis  SubphylumSarcodina (the amoebae)   Superclass Rhizopoda    Class Lobosea     Order Amoebida            Entamoeba histolytica Amebiasis PhylumApicomplexa    Class Sporozoea     Subclass Coccidia      OrderEucoccidiorida       Suborder Eimeriorina        Family Eimeriina           Isospora belli Isospora Infection        Family Sarcocystidae        Toxoplasma gondii (toxoplasmosis) Toxoplasmosis        FamilyCryptosporidiidae         Cryptosporidium parvum (cryptosporidosis)Cryptosporidiosis            Cyclospora cayetanensis Cyclosporiasis    Subclass Piroplasmasina        Family Babesiidae            Babesiaeg Babesia microti Babesia Infection            and Babesia divergensPhylum Ciliophora (the ciliates)    Class Litostomatea      OrderVestibuliferida            Balantidium coli Balantidium Infection PhylumPlathyhelminthes (the flatworms)    Class Trematoda     Subclass Digenea(the digenetic trematodes)      Order Echinostomatiformes        FamilyFasciolidea            Fasciola hepatica Fascicliasis           Fasciolopsis buski Fasciolopsiasis      Order Strigeiformes       Family Schistosomatidae            Schistosoma mansoni, S.haematobium, Schistosomiasis            and S. japonicum      OrderOpisthorchiformes        Family Opisthorchiidae            Clonorchissinensis Clonorchis Infection        Family Heterophyidae           Heterophyes heterophyes Heterophyes Infection      OrderPlagiorchiformes       Suborder Plagiorchiata        FamilyDicrocoeliidae         Platynostomum sp. Malaria       SuborderTroglotremata        Family Troglotrematidae            ParagonimusParagonimiasis    Class Cestoidea     Subclass Eucestoda (the tapeworms)     Order Cyclophyllidea            Dipylidium caninum DipylidiumInfection            Echinoccus multiloclaris Alveolar Hydatid Disease           Hymenolepis nana Hymenolepiasis            Taenia saginataTaeniasis, Cysticerosis      Order Proteocephalata           Opisthorohis viverrini Opisthorchis Infection      OrderPseudophyllidea            Diphyllobothrium latum DiphyllobothriumInfection Phylum Nematoda (the roundworms)    Class Aphasmida (=Enoplea)     Order Trichurida        Family Capillaridae            Capillariaphilippinensis Capillaria Infection        Family Trichinellidae           Trichinella Trichinellosis/Trichinosis        FamilyTrichuridae            Trichuris trichiura Trichuriasis    ClassRhabditae      Order Rhabditidae            Strongyloides stercoralisStrongyloidiasis      Order Strongylida        Family Ancylostomidae           Ancylostoma duodenale Hookworm Infection           Angiostrongylus cantonensis Angiostrongyliasis      OrderAscaridida            Ascaris Ascaris Infection            Toxocaracanis, T. cati Roundworm Infection, Intestinal            BaylisascarisBaylisascaris Infection            Anisakis simplex and Anisakiasis           Pseudoterranova decipiens      Order Oxyurida           Enterobius vermicularis Pinworm Infection      OrderSpirurida       Suborder Spirurina        Family Onchocercidae        Onchocerca volvulus (onchocerciasis, riverblindness)Onchocerciasis       Suborder Camallanina        Family Dracunculidae           Dracunculus medinensis Guinea Worm Disease Phylum Arthropoda Subphylum Crustacea    Class insecta      Order Anoplura           Pediculus humanus capitis Pediculosis  Subphylum Chelicerata   Class Arachnida      Order Acari       Suborder Astigmata        Sarcoptes scabiei Scabies

In a related embodiment, the present invention provides for animmunogenic composition and method of use, where the immunogeniccomposition comprises a PIKA adjuvant together with an allergy antigen(“allergen”) or vaccine where the source of the antigen or vaccine isderived from or produced to emulate a pathogen from a human or animalallergy sources including; plants, animals, fungi, insects, food, drugs,dust, and mites and the like.

Allergens include but are not limited to environmental aeroallergens;plant pollens such as ragweed/hayfever; weed pollen allergens; grasspollen allergens; Johnson grass; tree pollen allergens; ryegrass;arachnid allergens, such as house dust mite allergens (e.g., Der p I,Der f I, etc.); storage mite allergens; Japanese cedar pollen/hay fever;mold spore allergens; animal allergens (e.g., dog, guinea pig, hamster,gerbil, rat, mouse, etc., allergens); food allergens (e.g., allergens ofcrustaceans; nuts, such as peanuts; citrus fruits); insect allergens;venoms: (Hymenoptera, yellow jacket, honey bee, wasp, hornet, fire ant);Other environmental insect allergens from cockroaches, fleas,mosquitoes, etc.; bacterial allergens such as streptococcal antigens;parasite allergens such as Ascaris antigen; viral antigens; fungalspores; drug allergens; antibiotics; penicillins and related compounds;other antibiotics; whole proteins such as hormones (insulin), enzymes(streptokinase); all drugs and their metabolites capable of acting asincomplete antigens or haptens; industrial chemicals and metabolitescapable of acting as haptens and functioning as allergens (e.g., theacid anhydrides (such as trimellitic anhydride) and the isocyanates(such as toluene diisocyanate)); occupational allergens such as flour(e.g., allergens causing Baker's asthma), castor bean, coffee bean, andindustrial chemicals described above; flea allergens; and human proteinsin non-human animals.

Allergens include but are not limited to cells, cell extracts, proteins,polypeptides, peptides, polysaccharides, polysaccharide conjugates,peptide and non-peptide mimics of polysaccharides and other molecules,small molecules, lipids, glycolipids, and carbohydrates.

Examples of specific natural, animal and plant allergens include but arenot limited to proteins specific to the following genuses: Canine (Canisfamiliaris); Dermatophagoides (e.g. Dermatophagoides farinae); Felis(Felis domesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g.Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeriajaponica); Alternaria (Alternaria alternata); Alder; Alnus (Alnusgultinoasa); Betula (Betula verrucosa); Quercus (Quercus alba); Olea(Olea europa); Artemisia (Artemisia vulgaris); Plantago (e.g. Plantagolanceolata); Parietaria (e.g. Parietaria officinalis or Parietariajudaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apismultiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressusarizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperussabinoides, Juniperus virginiana, Juniperus communis and Juniperusashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g. Chamaecyparisobtusa); Periplaneta (e.g. Periplaneta americana); Agropyron (e.g.Agropyron repens); Secale (e.g. Secale cereale); Triticum (e.g. Triticumaestivum); Dactylis (e.g. Dactylis glomerata); Festuca (e.g. Festucaelatior); Poa (e.g. Poapratensis or Poa compressa); Avena (e.g. Avenasativa); Holcus (e.g. Holcus lanatus); Anthoxanthum (e.g. Anthoxanthumodoratum); Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g.Agrostis alba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalarisarundinacea); Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghumhalepensis); and Bromus (e.g. Bromus inermis).

In a related embodiment, the present invention provides for apolynucleotide adjuvant composition and -method of use where theimmunogenic composition comprises a PIKA adjuvant together with anautoimmune antigen or vaccine.

In a related embodiment, the present invention provides for animmunogenic composition and method of use, where the immunogeniccomposition comprises the PIKA adjuvant alone or together with a cancerantigen, wherein exemplary antigens include but are not limited toantigens of one or more of the cancers described in Table 5.

TABLE 5 Cancers Cancer Taxonomy and Diseases 1 Bone 2   Ewing's Familyof Tumors 3   Osteosarcoma 4 Brain 5   Brain Tumor 6   Brain Stem Glioma7   Cerebellar Astrocytoma 8   Cerebral Astrocytoma/Malignant Glioma 9  Ependymoma 10   Medulloblastoma 11   Supratentorial PrimitiveNeuroectodermal Tumors and   Pineoblastoma 12   Visual Pathway andHypothalamic Glioma 13 14 Breast 15   Breast Cancer 16Digestive/Gastrointestinal 17   Anal Cancer 18   Bile Duct Cancer,Extrahepatic 19   Carcinoid Tumor, Gastrointestinal 20   Colon Cancer 21  Esophageal Cancer 22   Gallbladder Cancer 23   Liver Cancer 24  Pancreatic Cancer 25   Rectal Cancer 26   Small Intestine Cancer 27  Stomach (Gastric) Cancer 28 Endocrine 29   Adrenocortical Carcinoma 30  Carcinoid Tumor, Gastrointestinal 31   Islet Cell Carcinoma (EndocrinePancreas) 32   Parathyroid Cancer 33   Pheochromocytoma 34   PituitaryTumor 35   Thyroid Cancer 36 Eye 37   Melanoma, Intraocular 38  Retinoblastoma 39 Genitourinary 40   Bladder Cancer 41   Kidney (RenalCell) Cancer 42   Penile Cancer 43   Prostate Cancer 44   Renal Pelvisand Ureter Cancer, Transitional Cell 45   Testicular Cancer 46  Urethral Cancer 47   Wilms' Tumor and Other Childhood Kidney Tumors 48Germ Cell 49   Extracranial Germ Cell Tumor 50   Extragonadal Germ CellTumor 51   Ovarian Germ Cell Tumor 52   Testicular Cancer 53 Gynecologic54   Cervical Cancer 55   Endometrial Cancer 56   GestationalTrophoblastic Tumor 57   Ovarian Epithelial Cancer 58   Ovarian GermCell Tumor 59   Ovarian Low Malignant Potential Tumor 60   UterineSarcoma 61   Vaginal Cancer 62   Vulvar Cancer 63 64 Head and Neck 65  Hypopharyngeal Cancer 66   Laryngeal Cancer 67   Lip and Oral CavityCancer 68   Metastatic Squamous Neck Cancer with Occult Primary 69  Nasopharyngeal Cancer 70   Oropharyngeal Cancer 71   Paranasal Sinusand Nasal Cavity Cancer 72   Parathyroid Cancer 73   Salivary GlandCancer 74 Hematologic/Blood 75  Leukemia 76   Acute LymphoblasticLeukemia 77   Acute Myeloid Leukemia 78   Chronic Lymphocytic Leukemia79   Chronic Myelogenous Leukemia 80   Hairy Cell Leukemia 81  Lymphoma82   AIDS-Related Lymphoma 83   Cutaneous T-Cell Lymphoma 84   Hodgkin'sLymphoma 85   Mycosis Fungoides 86   Non-Hodgkin's Lymphoma 87   PrimaryCentral Nervous System Lymphoma 88   Sezary Syndrome 89   T-CellLymphoma, Cutaneous 90   Waldenstrom's Macroglobulinemia 91  Other 92  Chronic Myeloproliferative Disorders 93   Multiple Myeloma/Plasma CellNeoplasm 94   Myelodysplastic Syndromes 95  Myelodysplastic/Myeloproliferative Diseases 96  Lung 97   Non-SmallCell Lung Cancer 98   Small Cell Lung Cancer 99  Musculoskeletal 100  Ewing's Family of Tumors 101   Osteosarcoma/Malignant FibrousHistiocytoma of Bone 102   Rhabdomyosarcoma 103   Soft Tissue Sarcoma104   Uterine Sarcoma 105  Neurologic 106   Brain Tumor 107   Brain StemGlioma 108   Cerebellar Astrocytoma 109   Cerebral Astrocytoma/MalignantGlioma 110   Ependymoma 111   Medulloblastoma 112   SupratentorialPrimitive Neuroectodermal Tumors and Pineoblastoma 113   Visual Pathwayand Hypothalamic Glioma 114   Neuroblastoma 115   Pituitary Tumor 116  Primary Central Nervous System Lymphoma 117  Respiratory/Thoracic 118  Lung Cancer, Non-Small Cell 119   Lung Cancer, Small Cell 120  Malignant Mesothelioma 121   Thymoma and Thymic Carcinoma 122  Skin123   Cutaneous T-Cell Lymphoma 124   Kaposi's Sarcoma 125   Melanoma126   Merkel Cell Carcinoma 127   Skin Cancer

In a related embodiment the source of the cancer antigen may be: 1)Viral proteins—for example hepatitis B virus (HBV), Epstein-Barr virus(EBV) and human papillomavirus (HPV)—are important in the development ofhepatocellular carcinoma, lymphoma, and cervical cancer, respectively;2). whole cancer cells that may be inactivated and/or nonpurified and/orsemi-purified extract of these cells; 3). tumor-associated antigens(TAAs) such as the tumor specific oncogenic proteins, glycosylatedproteins, gangliosides, glycolipide, mucins, peptide, carbohydrates andanti-idiotype monoclonal antibodies.

In a related embodiment, the use of the immunogenic compositioncomprising the polynucleotide adjuvant may be for the treatment ofcancer tumors through the prevention of further growth of existingcancers, the prevention of the recurrence of treated cancers, or theelimination of cancer cells not killed by prior treatments. Thetreatment may be administered prior to, in conjunction with, or postother therapies provided to the individual and thus may form part of anoverall combination therapy to treat the cancer.

In a related embodiment the cancer vaccine provides for therapiescapable of inducing tumor specific immune responses against both aprimary tumor and metastases. In addition, the induction of a strongimmunity may lead to the establishment of immune memory, therebyreducing or inhibiting tumor recurrence. The cancer vaccine may inducespecific antibodies against tumor-associated surface antigens andpreferably to induce cellular immune response with preferably a biastoward a Th1 immune response.

Any of a variety of known tumor-specific antigens or tumor-associatedantigens (TAA) can be included in a subject immunogenic composition. Theentire TAA may be, but need not be, used. Instead, a portion of a TAA,e.g., an epitope, may be used. Tumor-associated antigens (orepitope-containing fragments thereof) which may be used into YFVinclude, but are not limited to, MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2,high molecular weight melanoma-associated antigen MAA, GD2,carcinoembryonic antigen (CEA), TAG-72, ovarian-associated antigensOV-TL3 and MOV18, TUAN, alpha-feto protein (AFP), OFP, CA-125, CA-50,CA-19-9, renal tumor-associated antigen G250, EGP-40 (also known asEpCAM), S100 (malignant melanoma-associated antigen), p53, and p21ras. Asynthetic analog of any TAA (or epitope thereof), including any of theforegoing, may be used. Furthermore, combinations of one or more TAAs(or epitopes thereof) may be included in the composition.

In some embodiments, a subject immunogenic composition comprises apolynucleotide adjuvant, and at least two different antigens, e.g., insome embodiments, a subject immunogenic composition comprises twoantigens, three antigens, four antigens, five antigens, or more thanfive antigens.

Additional agents

In some embodiments, a subject immunogenic composition comprises, inaddition to a PIKA adjuvant and an antigen, one or more additionalagents, e.g., immunomodulatory agents, carriers, and the like.

In an embodiment of particular interest, the present invention providesfor an immunogenic composition and method of use, where the immunogeniccomposition comprises the PIKA adjuvant, an antigen or vaccine togetherwith another immunomodulating substance, including adjuvants, wheresuitable immunomodulating substances include, but are not limited to: analuminum composition such as aluminum hydroxide; oil-in-water emulsionscompositions or emulsions comprising an immunogenic substances,including Complete Freund's Adjuvant; an oil-in-water emulsioncontaining dried, heat-killed Mycobacterium tuberculosis organisms;Incomplete Freund's Adjuvant; emulsions including mycobacterial cellwall components; emulsions including squalene (MF-59); detoxifiedendotoxins, lipid A derivatives including monophosphoryl lipidA-microbial (MPL); haptens; nitrocellulose-absorbed protein; saponinsincluding particulate immunomodulators isolated from the bark ofQuillaja Saponoria for example QS21; endogenous human immunomodulators;bacterial derived adjuvants including unmethylated CpG dinucleotides;oligodeoxynucleotides (e.g., synthetic oligonucleotides) containingunmethylated CpG dinucleotides; liposomes (e.g., liposomes made ofbiodegradable materials such as phospholipids); biodegradable polymermicrospheres (e.g., microspheres made from a variety of polymers such aspolylactic-co-glycolic acid (PLGA), polyphosphazene and polyanhydrides);Interleukin-2; Bacillus Calmette Guerin; Granulocyte Monocyte-ColonyStimulating Factor; Montanide ISA-51; Keyhole limpet hemocyanin; DNA;proteins; encapsulated antigens; immune stimulating complexes (ISCOM's);cholera toxin, choleral toxin derivatives; zonula occludens toxin;escherichia coli heat-labile enterotoxin; labile toxin, labile toxinderivatives; pertussis toxin, pertussis toxin derivatives; muramyldipeptide derivatives; seppic series of montanide adjuvants;poly-di(carboxylatophenoky)phosphazene and leishmania elongation factor.

When the subject immunogenic composition is administered in conjunctionwith another adjuvant, the polynucleotide adjuvant can be administeredbefore and/or after, and/or simultaneously with the other adjuvant. Forexample the polynucleotide adjuvant may be administered with the initialadministration of the antigen, followed by a boost dose of vaccinecomprising either or both of the adjuvants. Alternatively the initialdose of vaccine administered may exclude the polynucleotide adjuvantsbut an immunogenic substance comprising the polynucleotide adjuvant issubsequently administered to the patient.

In certain embodiments the subject immunogenic composition may beadministered with cytokines or other co-stimulatory molecules forexample: IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15

In a related embodiment the present invention provides for animmunogenic substance comprising a PIKA adjuvant, an antigenic substanceor substances, plus a suitable carrier. The carrier may be for examplean oil-and-water emulsion, suspension, a lipid vehicle, aluminum salt,cochleates, ISCOMs, liposomes, live bacterial vectors, live viralvectors, microspheres, nucleic acid vaccines, polymers, polymer rings,sodium fluoride, transgenic plants, virosomes, virus like particles, andother delivery vehicles known in the art.

The polynucleotide adjuvant may be directly administered to the subjector may be administered in conjunction with a delivery complex. Where thedelivery complex is a substance associated with a targeting means e.g. amolecule that results in higher affinity binding to target cell such asdendritic cell surfaces and/or increased cellular uptake by targetcells. Examples of delivery complexes include but are not limited to;nucleic acid delivery acids associated with: a sterol (e.g.cholesterol), a lipid (e.g. cationic lipid, virosome or liposome), or atarget cell specific binding agent (e.g. a ligand recognized by a targetcell specific receptor). Preferred complexes may be sufficiently stablein vivo to prevent significant uncoupling prior to internalization bythe target cell. However, the complex may be cleavable under appropriateconditions within the cell.

In one embodiment of interest, the composition comprising PIKA adjuvantdoes not include poly-L-lysine or a derivative thereof.

Kits

In certain embodiments, the invention provides a kit comprising asubject immunogenic composition. In certain embodiments, the inventionprovides a kit comprising a PIKA adjuvant and an antigen in separateformulations.

In a related embodiment, the invention provides for a kit comprising thepolynucleotide adjuvant and an immunogenic compound where theimmunogenic substance is an antigen.

In some embodiments, a subject kit comprises a subject immunogeniccomposition in a sterile liquid (e.g., aqueous) formulation, where theformulation is sterile, and is provided in a sterile container, asterile vial, or a sterile syringe.

In some embodiments, a subject kit comprises a subject immunogeniccomposition formulated for injection. In some embodiments, a subject kitcomprises a subject immunogenic composition in a sterile liquidformulation, contained within a sterile syringe; and a needle. In someembodiments, a subject kit comprises a subject immunogenic compositionin a sterile liquid formulation in a unit dosage amount (e.g., a singledose), contained within a sterile syringe; and a needle.

In some embodiments, a subject kit comprises a subject immunogeniccomposition, lyophilized and in a sterile container; and a containercomprising a sterile liquid for reconstitution of the lyophilizedcomposition. In some embodiments, the kit further comprises instructionsfor reconstitution of the lyophilized composition.

In some embodiments a subject kit comprises an immunogenic compositionformulated for administration rectally, vaginally, nasally, orally(including inhalation), opthamalically, topically, pulmonary, ocularlyor transdermally and an appropriate delivery device for example,inhaler, suppository, applicator or the like,

A subject kit in some embodiments will further include instructions foruse, including e.g., dosage amounts and dosage frequencies. Instructionsare in some embodiments printed directly on the kit. In otherembodiments, instructions are printed material provided as a packageinsert. Instructions can also be provided in other media, e.g.,electronically in digital or analog form, e.g., on an audio cassette, anaudio tape, a compact disc, a digital versatile disk, and the like.

Formulations

A subject immunogenic composition is provided in any of a variety offormulations. For example, a subject immunogenic composition may beprepared as an injectable, dry power, liquid solution, for example:aqueous or saline solution or as: a suspension, cream, emulsion, tablet,coated tablet, microcapsule, suppository, drops, pill, granules, dragee,capsule, gel, syrup or slurry. The preparation of formulations of adesired immunogenic composition is generally described in Vaccine 4^(th)Edition by Stanley A Plotkin et al., W.B. Saunders Company; 4th edition2003. Suitable formulations are also described in, e.g., A. Gennaro(2000) “Remington: The Science and Practice of Pharmacy,” 20^(th)edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Formsand Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7^(th) ed.,Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed. Amer.Pharmaceutical Assoc.; Methods in Molecular Medicine, Vol. 87: VaccineProtocols, 2nd edition (2003), Humana Press; Mucosal Vaccines (1996),Kiyono et al., eds., Academic Press; and Vaccine Adjuvants: PreparationMethods and Research Protocols (2000) D. T. O'Hagan, Humana Press.

A subject immunogenic composition may be microencapsulated,encochleated, coated onto microscopic gold partiles, contained inliposomes, nebulized aerosols, pellets for implantation into the skin,or dried onto a sharp object (e.g., a needle) to be scratched into theskin.

In a further embodiment the subject immunogenic substance may bedelivered alone or in conjunction with a dispersion system. In someembodiments the dispersion system is selected from the group consistingof for example: macromolecular complexes, nanocapsules, microspheres,beads and lipid based systems. Lipid based systems optionally includeoil-in-water emulsions, micelles, mixed micelles or liposomes.

In certain embodiments a subject immunogenic composition comprising thePIKA adjuvant is in the form of a pharmaceutically acceptable solution,which may routinely contain pharmaceutically acceptable concentrationsof salt, buffering agents, preservatives, compatible carriers, adjuvantsand optionally other therapeutic ingredients. The composition maycontain additives for example: disintegrants, binders, coating agents,swelling agents, lubricants, flavorings, sweeteners or solubilizers andthe like.

In certain embodiments a subject immunogenic composition comprising thePIKA adjuvant is administered in its neat for or in the form of apharmaceutically acceptable salt.

The immunogenic composition of the present invention may be employed insuch forms, both sterile and non-sterile, such as capsules, liquidsolutions, liquid drops, emulsions, suspensions, elixirs, creams,suppositories, gels, soft capsules, sprays, inhalants, aerosols,powders, tablets, coated tablets, lozenges, microcapsules,suppositories, dragees, syrups, slurries, granules, enemas or pills. Anyinert carrier can be used, such as saline, or phosphate buffered saline,stabilizers, propellants, encased in gelatin capsule or in amicrocapsule or vector that aids administration or any such carrier inwhich the compounds used in the method of the present invention havesuitable solubility properties for use in the methods of the presentinvention.

In certain embodiments, the PIKA adjuvant composition and an immunogeniccomposition comprising the PIKA adjuvant and antigenic compound isfreeze-dried (lyophilized) for long term stability and storage in asolid form. The freeze-dried method is known to those skilled in theart.

In one aspect of particular interest, the invention provides for anadjuvant composition or immunogenic composition wherein the immunogeniccomposition, or the adjuvant composition contained in the immunogeniccomposition, is in a solid or liquid form or in solution or insuspension or in emulsion.

A subject immunogenic composition may be administered to an individualby means of a pharmaceutical delivery system for the inhalation route(oral, intratracheal, intranasal). Thus, a subject immunogeniccomposition may be formulated in a form suitable for administration byinhalation. The pharmaceutical delivery system is one that is suitablefor respiratory therapy by topical administration of a subject bacterialcomposition to mucosal linings of the bronchi. This invention canutilize a system that depends on the power of a compressed gas to expelthe bacteria from a container. An aerosol or pressurized package can beemployed for this purpose.

As used herein, the term “aerosol” is used in its conventional sense asreferring to very fine liquid or solid particles carries by a propellantgas under pressure to a site of therapeutic application. When apharmaceutical aerosol is employed in this invention, the aerosolcontains the immunogenic composition, which can be dissolved, suspended,or emulsified in a mixture of a fluid carrier and a propellant. Theaerosol can be in the form of a solution, suspension, emulsion, powder,or semi-solid preparation. Aerosols employed in the present inventionare intended for administration as fine, solid particles or as liquidmists via the respiratory tract of a subject. Various types ofpropellants known to one of skill in the art can be utilized. Examplesof suitable propellants include, but are not limited to, hydrocarbons orother suitable gas. In the case of the pressurized aerosol, the dosageunit may be determined by providing a value to deliver a metered amount.

There are several different types of inhalation methodologies which canbe employed in connection with the present invention. A subjectimmunogenic composition can be formulated in basically three differenttypes of formulations for inhalation. First, a subject immunogeniccomposition can be formulated with low boiling point propellants. Suchformulations are generally administered by conventional meter doseinhalers (MDI's). However, conventional MDI's can be modified so as toincrease the ability to obtain repeatable dosing by utilizing technologywhich measures the inspiratory volume and flow rate of the subject asdiscussed within U.S. Pat. Nos. 5,404,871 and 5,542,410.

Alternatively, a subject immunogenic composition can be formulated inaqueous or ethanolic solutions and delivered by conventional nebulizers.In some embodiments, such solution formulations are aerosolized usingdevices and systems such as disclosed within U.S. Pat. Nos. 5,497,763;5,544,646; 5,718,222; and 5,660,166.

Furthermore, a subject immunogenic composition can be formulated intodry powder formulations. Such formulations can be administered by simplyinhaling the dry powder formulation after creating an aerosol mist ofthe powder. Technology for carrying such out is described within U.S.Pat. No. 5,775,320 and U.S. Pat. No. 5,740,794. Formulations suitablefor intranasal administration include nasal sprays, nasal drops, aerosolformulations; and the like.

In some embodiments, a subject immunogenic composition is formulated asa sustained release (e.g. a controlled release formulation). Forexample, in some embodiments, a subject immunogenic composition isformulated into pellets or cylinders and implanted intramuscularly orsubcutaneously as depot injections or as implants. Such implants willgenerally employ known inert materials such as biodegradable polymers.Injectable depot forms are made by forming microencapsule matrices of asubject immunogenic composition in biodegradable polymers such aspolylactide-polyglycolide. Examples of other suitable biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are also prepared by entrapping the compositionin liposomes or microemulsions which are compatible with body tissue.Delivery release systems also include the following examples: polymerbased systems, microcapsules, lipids, hydrogel release systems, sylasticsystems, peptide systems, peptide based systems, wax coatings,compressed tablets, partially fused implants, Other forms of sustainedrelease are known by those skilled in the art.

Methods

In one aspect of particular interest, the invention provides for amethod for eliciting and/or enhancing immune responses to an antigeniccompound, comprising administering to a host a subject immunogeniccomposition. In some embodiments, the host is a human. In otherembodiments, the host is a non-human animal, e.g., a non-human mammal,an avian species, etc.

In certain embodiments, the polynucleotide adjuvant composition can beused in the context of a vaccine. Optionally, the vaccine compositioncontains additional adjuvants. Vaccines classes included areanti-infectious diseases, anti cancer, anti-allergy and anti-autoimmunediseases.

Furthermore, the present invention provides a method for enhancingimmune responses to an antigenic compound by administering to a host asubject immunogenic composition. The host can be a human being ornon-human animal. In certain embodiments the adjuvant is administeredtogether with the antigen. The further embodiments the adjuvant isadministered prior to or post the administration of the antigen.

A subject immunogenic composition is in some embodiments deliveredparenterally by injection, such as intramuscular, intraperitoneal,intravenous, subcutaneous or intradermal injection. In other embodimentsthe immunogenic composition is administered intradermally in ways otherthan by injection for example, without breaching the epithelial barrierby mechanical means. In other embodiments, the immunogenic compositionis delivered rectally, vaginally, nasally, orally (includinginhalation), opthamalically, ocularly, topically, pulmonary ortransdermally.

The subject may be exposed to the antigen through environmental contactand therefore at risk of developing for example, an allergic reaction,an infectious disease, autoimmune disease or a cancer. In otherembodiments the subject has for example an infectious disease,autoimmune disease, a cancer or allergy as a result of prior exposure toan antigen through environmental contact.

In certain embodiments, when the mode of administration of theimmunogenic composition comprising a polynucleotide adjuvant is for thetreatment of cancer tumors, the delivery is by injection directly intothe tumor, or adjacent to the tumor. In some embodiments, theimmunogenic composition is delivered evenly over or throughout the tumorto enhance the biodistribution and hence enhance the therapeuticbenefit.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous andintraperitoneal administration. In this connection, sterile aqueousmedia which can be employed will be known to those of skill in the artin light of the present disclosure. Exemplary injection media which canbe used in the present invention include a buffer with or withoutdispersing agents and/or preservatives, and edible oil, mineral oil, codliver oil, squalene, mono-, di- or triglyceride, and a mixture thereof.

A subject immunogenic composition is administered in an “effectiveamount” that is, an amount of a subject immunogenic composition that iseffective in a selected route of administration to elicit, induce, orenhance an immune response. In some embodiments, an immune response iselicited to antigens produced by a pathogenic microorganism. In someembodiments, the amount of a subject immunogenic composition iseffective to limit an infection, and/or to eradicate an infection,and/or to reduce a symptom associated with infection, by a pathogenicorganism.

For example, in some embodiments, administration of a subjectimmunogenic composition to an individual is effective to treat aninfectious disease, where treating an infectious disease, encompassesone or more of reducing the number of pathogenic agents in theindividual (e.g., reducing viral load, reducing bacterial load, reducingthe number of protozoa, reducing the number of helminths) and/orreducing a parameter associated with the infectious disease, including,but not limited to, reduction of a level of a product produced by theinfectious agent (e.g., a toxin, an antigen, and the like); and reducingan undesired physiological response to the infectious agent (e.g.,fever, tissue edema, and the like).

The exact amount of such compositions required will vary from subject tosubject, depending on the species, age, weight, and general conditionsof the subject, the severity of the disease, infection, or conditionthat is being treated or prevented, the particular compound used, itsmode administration, and the like. An appropriate amount may bedetermined by one of ordinary skill in the art using only routineexperimentation given the teachings herein. Following an initialadministration, subjects may receive one or several boosterimmunizations adequately spaced.

In some embodiments, serial doses of a subject immunogenic compositionare administered. In these embodiments, the first dose of a subjectimmunogenic composition may be as a result of administering a vaccine.The second dose of a subject immunogenic composition is administered tothe individual after the individual has been immunologically primed byexposure to the first dose. The booster may be administered days, weeksor months after the initial immunization, depending upon the patient'sresponse and condition. For example, the booster dose is administeredfrom about 2 days to about 12 months after the initial dose, e.g., fromabout 2 days to about 7 days, from about 1 week to about 2 weeks, fromabout 2 weeks to about 4 weeks, from about 4 weeks to about 8 weeks,from about 8 weeks to about 6 months, or from about 6 months to about 12months after the initial dose. The present invention furthercontemplates the use of a third, fourth, fifth, sixth or subsequentbooster immunization, using, e.g., a third, fourth, fifth, sixth, orsubsequent dose.

In certain embodiments the means of administration may comprise acombination of alternative routes, for example: systemicallyadministered dose (e.g. peritoneal, intra-muscular, subcutaneous orintradermal administration) may be followed by mucosally delivered dose(e.g. intranasal, inhalation) or vice versa.

In certain embodiments the polynucleotide adjuvant may be administeredwith either the first dose of antigen administered or any of thesubsequent doses administered or all doses administered to the patient.At least one of the doses administered as part of the overall protocolwould comprise the PIKA adjuvant.

In certain embodiments the composition of the administered immunogeniccomposition may vary between the original administration and the boostand/or between booster doses. By way of an example the original doseadministered may comprise a DNA vaccine while the booster dose is in theform of a recombinant protein vaccine. At least one of the dosesadministered as part of the overall protocol would comprise the PIKAadjuvant.

Whether an antibody response to an antigen has been induced or enhancedin an individual is readily determined using standard assays. Forexample, immunological assays such as enzyme-linked immunosorbent assays(ELISA), radioimmunoassay (RIA), immunoprecipitation assays, and proteinblot (“Western” blot) assays; and neutralization assays (e.g.,neutralization of viral infectivity in an in vitro or in vivo assay);can be used to detect the presence of antibody specific for a microbialantigen in a bodily fluid or other biological sample, e.g., the serum,secretion, or other fluid, of an individual.

Whether a CD4 immune response to an antigen has been induced in anindividual is readily determined using standard assays, e.g.,fluorescence-activated cell sorting (FACS) (see, e.g., Waldrop et al.(1997) J. Clin. Invest. 99:1739-1750); intracellular cytokine assaysthat detect production of cytokines following antigen stimulation (see,e.g., Suni et al. (1998) J. Immunol. Methods 212:89-98; Nomura et al.(2000) Cytometry 40:60-68; Ghanekar et al. (2001) Clin. Diagnostic Lab.Immunol. 8:628-631); MHC-peptide multimer staining assays, e.g., use ofdetectably labeled (e.g., fluorescently labeled) soluble MHC ClassII/peptide multimers (see, e.g., Bill and Kotzin (2002) Arthritis Res.4:261-265; Altman et al. (1996) Science 274:94-96; and Murali-Krishna etal. (1998) Immunity 8:177-187); enzyme-linked immunospot (ELISPOT)assays (see, e.g., Hutchings et al. (1989) J. Immunol. Methods 120:1-8;and Czerkinsky et al. (1983) J. Immunol. Methods 65:109-121); and thelike. As one non-limiting example of an intracellular cytokine assay,whole blood is stimulated with antigen and co-stimulating antibodies(e.g., anti-CD28, anti-CD49d) for 2 hours or more; Brefeldin A is addedto inhibit cytokine secretion; and the cells are processed for FACSanalysis, using fluorescently labeled antibodies to CD4 and to cytokinessuch as TNF-a, IFN-γ and IL-2.

Whether an antigen-specific CD8 (e.g., cytotoxic T cell; “CTL”) responseis induced to an antigen (e.g., to a pathogen) can be determined usingany of a number of assays known in the art, including, but not limitedto, measuring specific lysis by CTL of target cells expressing theantigen on their surface, which target cells have incorporated adetectable label which is released from target cells upon lysis, and canbe measured, using, e.g., a ⁵¹Cr-release assay; a lanthanidefluorescence-based cytolysis assay; and the like.

Subjects Suitable for Treatment

Subjects suitable for treatment with a subject method of inducing animmune response to a microbial pathogen, and methods of treating orpreventing an infection with a microbial pathogen, include individualswho have been infected with a pathogenic microorganism; individuals whoare susceptible to infection by a pathogenic microorganism, but who havenot yet been infected; and individuals who are at risk of becominginfected with a pathogenic microorganism, but who have not yet beeninfected. Suitable subjects include infants, children, adolescents, andadults.

Subjects suitable for treatment with a subject method of inducing animmune response to a microbial pathogen, and methods of treating orlimiting an infection with a microbial pathogen, include pediatrictarget population, e.g., individuals between about 1 year of age andabout 17 years of age, including infants (e.g., from about 1 month oldto about 1 year old); children (e.g., from about 1 year old to about 12years old); and adolescents (e.g., from about 13 years old to about 17years old).

Subjects suitable for treatment with a subject method of inducing animmune response to a microbial pathogen, and methods of treating orlimiting an infection with a microbial pathogen, include neonates, e.g.,an individual (e.g., a human neonate) from one day to about 14 days old,e.g., from about 1 day to about 2 days old, from about two days to about10 days old, or from about 10 days to about 14 days old.

In a particular embodiment, the subject is a human child about ten yearsor younger, e.g., about five years old or younger, and the immunogeniccompositions are administered at any one or more of the following times:two weeks, one month, 2 months, 3 months, 4 months, 5 months, 6 months,7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 15months, 18 months, or 21 months after birth, or at 2 years, 3 years, 4years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years of age.In some embodiments, a subject immunogenic composition is administeredto an individual in the age range of from about 6 months to about 6years, where the individual receives a first dose at about 6 months ofage, and subsequent booster doses, e.g., 2-3 subsequent booster doses,at, e.g., 2 years of age, 4 years of age, and 6 years of age.

In a particular embodiment, the subject is a human adult from about 17years old to 49 years old. In some embodiments, the subject is anelderly human adult from 50 to 65 years old, 65 to 75 years old, 75 to85 years old or over 85 years old.

In some embodiments, a subject immunogenic composition is administeredto an individual shortly after contact (e.g., shortly after confirmed orsuspected contact) with an actual or potential source of the microbialpathogen, for example, an individual who is known to have or suspectedto have an infection with a microbial pathogen. For example, in someembodiments, a subject immunogenic composition is administered to anindividual within about 1 hour, within about 2 hours, within about 5hours, within about 8 hours, within about 12 hours, within about 18hours, within about 24 hours, within about 2 days, within about 4 days,within about 7 days, within about 2 weeks, or within about one monthafter contact with an individual who is known to have or suspected tohave an infection with a microbial pathogen.

In some embodiments, a subject immunogenic composition is administeredto an individual that is known or may be suspected of being a carrier ora microbial pathogen whether or not they are showing symptoms of theinfection.

Subjects suitable for treatment with a subject method of inducing animmune response to a microbial pathogen, and methods of treating orlimiting an infection with a microbial pathogen, include CD4⁺ Tcell-deficient individuals (“CD4⁺-deficient” individuals), e.g.,individuals who have lower than normal numbers of functional CD4⁺ Tlymphocytes. As used herein, the term “normal individual” refers to anindividual having CD4⁺ T lymphocyte levels and function(s) within thenormal range in the population, for humans, typically 600 to 1500 CD4⁺ Tlymphocytes per mm³ blood. CD4⁺-deficient individuals includeindividuals who have an acquired immunodeficiency, or a primaryimmunodeficiency. An acquired immunodeficiency may be a temporary CD4⁺deficiency, such as one caused by radiation therapy, or chemotherapy.

Also suitable for treatment with the methods of the invention areindividuals with healthy, intact immune systems, but who are at risk forbecoming CD4⁺ deficient (“at-risk” individuals). At-risk individualsinclude, but are not limited to, individuals who have a greaterlikelihood than the general population of becoming CD4⁺ deficient.Individuals at risk for becoming CD4⁺ deficient include, but are notlimited to, individuals at risk for HIV infection due to sexual activitywith HIV-infected individuals; intravenous drug users; individuals whomay have been exposed to HIV-infected blood, blood products, or otherHIV-contaminated body fluids; a baby who has passed through the birthcanal of an HIV-infected individual; babies who are being nursed byHIV-infected mothers; and the like.

Subjects suitable for treatment with a subject method for treatingcancer include individuals who have been infected with a carcinogenicsubstance, individuals who are susceptible to cancer but who have notyet been diagnosed with cancer; and individuals who are at risk ofcontracting cancer, but who have not yet been diagnosed with cancer.Suitable subjects include infants, children, adolescents, and adults.

Subjects suitable for treatment with a subject method for treatingcancer include individuals who have been diagnosed with cancer;individuals who were previously treated for cancer, e.g., bychemotherapy or radiotherapy, and who are being monitored for recurrenceof the cancer for which they were previously treated; and individualswho have undergone bone marrow transplantation or any other organtransplantation.

Subjects suitable for treatment with the formulations and methods of theinstant invention for treating allergy include any individual who hasbeen diagnosed as having an allergy. Subjects amenable to treatmentusing the methods and agents described herein include individuals whoare known to have allergic hypersensitivity to one or more allergens.Subjects amenable to treatment include those who have any of theabove-mentioned allergic disorders. Also amenable to treatment aresubjects that are at risk of having an allergic reaction to one or moreallergens. Also suitable are individuals who failed treatment with oneor more standard therapies for treating an allergic disorder.

Subjects suitable for treatment include individuals living inindustrialized nations; individuals living developing countries;individuals living in rural areas; individuals living in relativelyisolated areas; and the like.

The target population for a subject immunogenic composition will vary,depending on the microbial pathogen

The above disclosure generally describes the present invention. Thefollowing examples will be of assistance to the understanding of thepresent invention. These examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

EXAMPLES Example 1 PIKA In Combination with a Variety of AntigensInduces a Specific Immune Response

This example involves use of PIKA in combination with a variety ofantigens to elicit a specific immune response in vivo. The research wasconducted in a series of independent experiments with a common protocolthough using a different antigen each time. The antigens tested include:a recombinant protein hepatitis B surface antigen type adw, aninactivated split influenza vaccine (VAXIGRIP from Sanofi Pasteur), asynthesized HIV peptide antigen, a recombinant protein herpes simplexvirus type 2 gD antigen, recombinant protective anthrax protein antigen,inactivated whole virus avian influenza antigen strain H5N1 and aninactivated whole virus Severe Acute Respiratory Syndrome (SARS)inactivated antigen.

The protocol for the individual experiment involves the inoculation ofgroups of Balb/c mice, three mice per group, with compositions ofantigen alone, antigen with the PIKA adjuvant (a heterogeneouscomposition of PIKA molecules predominantly within a weight rangedistribution of about 66 kDa to 1,200 kDa), PIKA alone, a controlcomprising phosphate buffer solution (PBS).

Actual dosage quantities are provided for each antigen used. The micewere then given an identical booster vaccine ten to fourteen days afterthe initial injection. Ten to fourteen days after the booster injectiona blood sample was taken, the mice were then sacrificed and tissuesamples taken from the spleen. The results presented are the average ofthe test results of the individual mice within each group.

A suspension of spleen cells was prepared and a sample of the cellsuspension from each mouse was put into 6-12 wells of the ELISPOT plateand cultured. Each well of the ELISPOT plate contained 200 ul ofsplenocyte suspension, approximately 2×10⁵ to 1×10⁶ cells/well (seedetails in tables below). For each mouse's sample of culturedsplenocytes, half of wells containing the splenocytes were incubatedwith culture medium and the other half of wells were stimulated usingthe one of two different concentrations of particular antigen underevaluation. Plates are incubated at 37° C. for 20 hours inenvironmentally controlled conditions prior to final preparation andreading using a standard ELISPOT plate reader.

Standard ELISPOT tests, known to those skilled in the art, were used todetect the number of cells producing the cytokines IL-4, IL-2 and INF-γ.

Flow Cytometry analysis was used to detect INF-γ produced by CD4+ Tcells. The use of Fluorescence-Activated Cell Sorter (FACS) is wellknown by those skilled in the art. In brief solutions of splenocytes ata concentration of 2.5×10⁶ cells/ml were prepared and divided intoindividual tubes with 2 ml per sample. Samples stimulated with antigenwere then prepared and after incubation at 37 C in environmentallycontrolled conditions for 5 hours. The samples then were washed andstained prior to reading in a standard FACS reader.

Standard ELISA tests known to those skilled in the art were use todetect the titer of specific antibodies in blood serum taken from theanimal prior to sacrifice.

Example 1.1 Recombinant Hepatitis B Surface Antigen (HBSAg) adw

The results in table 6 below are the results of the ELISPOT testdetecting the presence number of cells producing INF-γ, IL-2 and IL-4using a recombinant protein hepatitis B surface antigen (HBsAg) typeadw. The data in the table 6 (see also FIGS. 1, 2 and 3) represent theELISPOT reading, the number of spot forming cells, that is, a directmeasure the number of cells producing cytokine.

The distinct increase in the number of spot forming cells with theaddition of the PIKA adjuvant (as compared with the antigen alone)demonstrates that the addition of the PIKA adjuvant to recombinanthepatitis B surface antigen enhances the expression of cytokines INF-γ,IL-2 and IL-4 by cultured spleen cells. The observed expression ofcytokines indicates an enhanced adaptive immune response of both ahumoral and cell mediated immunity induced by the presence of the PIKAadjuvant.

TABLE 6 ELISPOT detection of murine splenocytes producing cytokinesafter immunization with vaccines comprising PIKA and/or HBsAg adw Groupsof Mice Group 1 Group 2 Group 3 HBsAg 4 ug + HBsAg PIKA Group 4 PIKA 50ug 4 ug 100 ug PBS No. of cells producing 539 26 — 2 IFN-γ/1 × 10⁶splenocytes No. of cells producing IL-2/ 347 60 3 10 1 × 10⁶ splenocytesNo. of cells producing IL-4/ 210 82 — 1 1 × 10⁶ splenocytes Stimulationwith HBsAg 2.0 ug/ml Unit: Splenocyte spot forming cells

Results of the ELISA test on the blood sample taken prior to sacrifice(Table 7 below and FIG. 4) demonstrate that the presence of PIKAsignificantly enhances the immune response and measured by the titer ofspecific antibodies detected in the serum.

TABLE 7 ELISA detection of specific IgG titers from murine serum afterimmunization with vaccines comprising PIKA and/or HBsAg adw Groups ofMice Group 1 Group 2 Group 3 HBsAg 4 ug + HBsAg PIKA Group 4 PIKA 50 ug4 ug 100 ug PBS Optical density absorbance 2.057 0.323 0.084 0.08 405 nmat 400x dilution

The conclusion drawn is that the addition of the PIKA adjuvant enhancesthe overall immune response to HBsAg, in particular the specific immuneresponse, more particularly the adaptive immunity and more specificallypredominant Th1 bias immune response and promote the cell mediatedimmune response.

Example 1.2 VAXIGRIP (Sanofi Pasteur), Inactivated and PurifiedInfluenza Antigen Comprising; H1N1, H3N2 Like Strains andb/Shanghai5/36112002 Strain

The results in table 8 below are the results of the ELISPOT testdetecting the presence the number of cells producing INF-γ, IL-2 andIL-4 using VAXIGRIP vaccine a inactivated split human influenza vaccineproduced by Sanofi Pasteur. The data in the table 8 (see also FIGS. 5, 6and 7) represent the ELISPOT reading, the number of spot forming cells,that is, a direct measure of cytokine production.

The distinct increase in the number of spot forming cells with theaddition of the PIKA adjuvant (as compared with the antigen alone)demonstrates that the addition of the PIKA adjuvant to the influenzaantigen enhances the expression of cytokines INF-γ, IL-2 and IL-4 bycultured spleen cells. The observed expression of cytokines indicates anenhanced adaptive immune response of both a humoral and cell mediatedimmunity induced by the presence of the PIKA adjuvant.

TABLE 8 ELISPOT detection of murine splenocytes producing cytokinesafter immunization with vaccines comprising PIKA and/or inactivatedsplit influenza antigen Groups of mice Group 2 Group 3 Group 1 Flu 4.5ug + Flu 4.5 ug + Group 4 Group 5 Flu 4.5 ug PIKA 50 ug PIKA 100 ug PIKA100 ug PBS No. of cells producing IFN-γ Not  1 —  2 1 — per 2.5 × 10⁵splenocytes stimulated Stimulated 81 148  252 14  6 with flu No. ofcells producing IL-2 Not —  1 — 1 — per 2.5 × 10⁵ splenocytes stimulatedStimulated 23 85 122 1 1 with flu No. of cells producing IL-4 Not  5 10 6 4 6 per 2.5 × 10⁵ splenocytes stimulated Stimulated 25 38  51 9 9with flu Unit: Splenocyte spot forming cells

Results of the ELISA test on the blood sample taken prior to sacrifice(Table 9 below and FIG. 8) demonstrate that the presence of PIKAsignificantly enhances the immune response and measured by the titer ofspecific antibodies detected in the serum.

TABLE 9 ELISA detection of specific IgG titers from murine serum afterimmunization with vaccines comprising PIKA and/or inactivated splitinfluenza vaccines Groups of mice Group 2 Group 3 Group 1 Flu 4.5 ug +Flu 4.5 ug + Group 4 Group 5 Flu 4.5 ug PIKA 50 ug PIKA 100 ug PIKA 100ug PBS Optical density 1.381 1.952 2.630 0.083 0.080 absorbance 405 nmat 900x dilution

The conclusion drawn is that the addition of the PIKA adjuvant enhancesthe overall immune response to influenza antigen, in particular thespecific immune response, more particularly the adaptive immunity andmore specifically the cell mediated immune response.

The VAXIGRIP is an approved influenza vaccine recognized tosignificantly reduce the risk of contracting influenza. The addition ofPIKA enhances the level of cytokines produced thereby indicating that avaccine comprising VAXIGRIP and PIKA also elicits an immune responsethat significantly reduces the risk of contracting influenza.

Example 1.3 Synthesized HIV Peptide Antigen

The results in table 10 below are the results of the ELISPOT testdetecting the presence number of cells producing INF-γ, IL-2 and IL-4using an HIV peptide antigen. The data in the table 10 (see also FIGS.9, 10 and 11) represent the ELISPOT reading, the number of spot formingcells, that is, a direct measure of cytokine production.

The distinct increase in the number of spot forming cells with theaddition of the PIKA adjuvant (as compared with the antigen alone)demonstrates that the addition of the PIKA adjuvant to the HIV antigenenhances the expression of cytokines INF-γ, IL-2 and IL-4 by culturedspleen cells. The observed expression of cytokines indicates an enhancedadaptive immune response of both a humoral and cell mediated immunityinduced by the presence of the PIKA adjuvant.

TABLE 10 ELISPOT detection of murine splenocytes producing cytokinesafter immunization with vaccines comprising PIKA and/or HIV gp 120antigen Groups of mice Group 1 HIVgp 120 3 ug + Group 2 Group 3 Group 4PIKA 100 ug HIVgp 120 3 ug PIKA 100 ug NS No. of cells producing IFN-γ/Stimulate with 121 16 2 5 2.5 × 10⁵ splenocytes HIV gp120 4 ug/ml Notstimulated 1 1 2 1 No. of cells producing IL-2/ Stimulate with 166 56 45 2.5 × 10⁵ splenocytes HIV gp120 4 ug/ml Not stimulated 1 3 2 — No. ofcells producing IL-4/ Stimulate with 68 78 5 6 2.5 × 10⁵ splenocytes HIVgp120 4 ug/ml Not stimulated 11 5 5 3 Unit: Splenocyte spot formingcells

The results of the FACS analysis are presented in table 11 below (seealso FIG. 12). The presence of CD4+ T cells expressing INF-γ in only theformulations containing both PIKA and HIV antigen confirms theobservation that the adaptive immune response has reach a stage ofmaturity and that PIKA was instrumental in this process.

TABLE 11 FACS analysis of murine splenocytes after immunization withvaccines comprising PIKA and/or HIV gp 120 antigen Groups of Mice Group1 HIVgp 120 Group 2 Group 3 3 ug + HIVgp PIKA Group 4 PIKA 100 ug 120 3ug 100 ug NS % of CD4+ve cells 0.194% 0.017% 0.012% 0.014% producinginterferon-γ

The conclusion drawn is that the addition of the PIKA adjuvant with HIVantigen enhances the overall immune response, in particular the specificimmune response, more particularly the adaptive immunity and morespecifically the cell mediated immune response.

Example 1.4 Recombinant Anthrax Protective Antigen (rPA) from BacillusAnthracis

The results in table 12 below are the results of the ELISPOT testdetecting the presence of INF-γ, IL-2 and IL-4 using a recombinantanthrax. The data in the table 12 (see also FIGS. 13, 14 and 15)represent the ELISPOT reading, the number of spot forming cells, thatis, a direct measure of cytokine production.

The distinct increase in the number of spot forming cells with theaddition of the PIKA adjuvant (as compared with the antigen alone)demonstrates that the addition of the PIKA adjuvant to the anthraxantigen enhances the expression of cytokines INF-γ, IL-2 and IL-4 bycultured spleen cells. The observed expression of cytokines indicates anenhanced adaptive immune response of both a humoral and cell mediatedimmunity induced by the presence of the PIKA adjuvant.

TABLE 12 ELISPOT detection of murine splenocytes producing cytokinesafter immunization with vaccines comprising PIKA and/or anthrax rPAantigen Groups of mice Group 1 Anthrax rPA 3 ug + Group 2 Group 3 Group4 PIKA 100 ug Anthrax rPA 3 ug PIKA 100 ug NS No. of cells producingIFN-γ/ Stimulation with 283 9 2 5 2.5 × 10⁵ splenocytes anthrax rPA 4ug/ml Not stimulated 135 3 2 1 No. of cells producing IL-2/ Stimulationwith 134 12 3 4 2.5 × 10⁵ splenocytes anthrax rPA 4 ug/ml Not stimulated47 2 2 — No. of cells producing IL-4/ Stimulation with 61 10 6 3 2.5 ×10⁵ splenocytes anthrax rPA 4 ug/ml Not stimulated 9 4 5 3 Unit:Splenocyte spot forming cells

The results of the FACS analysis are presented in table 13 below (seealso FIG. 16). The presence of CD4+ T cells expressing INF-γ in only theformulations containing both PIKA and rPA antigen confirms theobservation that the adaptive immune response has reach a stage ofmaturity and that PIKA was instrumental in this process.

TABLE 13 FACS analysis of murine splenocytes after immunization withvaccines comprising PIKA and/or anthrax rPA antigen Groups of Mice Group1 Group 2 Anthrax Anthrax Group 3 rPA 3 ug + rPA PIKA Group 4 PIKA 100ug 3 ug 100 ug PBS % of CD4+ve cells producing 0.746% 0.003% 0.010%0.004% Interferon-γ

Results of the ELISA test on the blood sample taken prior to sacrifice(Table 14 below and FIGS. 16 and 17) demonstrate that the presence ofPIKA significantly enhances the immune response and measured by thetiter of specific antibodies detected in the serum.

A consistent result was observed 16 weeks after the initial vaccinationwhen blood samples from mice of the original groups A and B wereevaluated for the presence of specific antibodies using a standard ELISAtest. Again the presence of PIKA with the anthrax antigen induced asignificantly higher immune response as measured by the specificantibody titer in the serum.

TABLE 14 ELISA detection of specific IgG titers from murine serum afterimmunization with vaccines comprising PIKA and/or anthrax rPA antigenGroups of mice Group 1 Anthrax rPA 3 ug + Group 2 Group 3 Group 4 PIKA100 ug Anthrax rPA 3 ug PIKA 100 ug PBS Optical density absorbance 4weeks after 1.29 0.12 0.1 0.1 405 nm at 400x dilution immunizationOptical density absorbance 16 weeks after 1.03 0.09 405 nm at 300xdilution immunization

The conclusion drawn is that the addition of the PIKA adjuvant enhancesthe overall immune response to rPA, in particular the specific immuneresponse, more particularly the adaptive immunity and more specificallythe cell mediated immune response.

Example 1.5 Recombinant Herpes Simplex Virus 2 gD Antigen

The results in table 15 below are the results of the ELISPOT testdetecting the presence of INF-γ, IL-2 and IL-4 using a recombinantherpes simplex virus antigen. The data in the table 15 (see also FIGS.19, 20 and 21) represent the ELISPOT reading, the number of spot formingcells, that is, a direct measure of cytokine production.

The distinct increase in the number of spot forming cells with theaddition of the PIKA adjuvant (as compared with the antigen alone)demonstrates that the addition of the PIKA adjuvant to the herpessimplex virus antigen enhances the expression of cytokines INF-γ, IL-2and IL-4 by cultured spleen cells. The observed expression of cytokinesindicates an enhanced adaptive immune response of both a humoral andcell mediated immunity induced by the presence of the PIKA adjuvant.

TABLE 15 ELISPOT detection of murine splenocytes producing cytokinesafter immunization with vaccines comprising PIKA and/or HSV 2gD antigenGroups of mice Group 1 HSV2gD 3 ug + PIKA Group 2 Group 3 Group 4 100 ugHSV2gp 3 ug PIKA 100 ug PBS No. of cells producing IFN-γ/ Stimulationwith 266 135 2 12 2.5 × 10⁵ splenocytes HSV2gD 2.5 ug/ml Not stimulated2 2 —  1 No. of cells producing IL-2/ Stimulation with 153 57 4  4 2.5 ×10⁵ splenocytes HSV2gD 2.5 ug/ml Not stimulated 2 2 — — No. of cellsproducing IL-4/ Stimulation with 40 25 10  12 2.5 × 10⁵ splenocytesHSV2gD 2.5 ug/ml Not stimulated 17 13 11  17 Unit: Splenocyte spotforming cells

The results of the FACS analysis are presented in table 16 below (seealso FIG. 22). The presence of CD4+ T cells expressing INF-γ in only theformulations containing both PIKA and HSV antigen confirms theobservation that the adaptive immune response has reach a stage ofmaturity and that PIKA was instrumental in this process.

TABLE 16 FACS analysis of murine splenocytes after immunization withvaccines comprising PIKA and/or HSV 2gD antigen Groups of Mice Group 1HSV2gp Group 2 Group 3 3 ug + PIKA HSV2gp PIKA Group 4 100 ug 3 ug 100ug NS % of CD4+ve cells 0.436% 0.056% 0.009% 0.012% producinginterferon-γ

Results of the ELISA test on the blood sample taken prior to sacrifice(Table 17 below and FIG. 23) demonstrate that the presence of PIKAsignificantly enhances the immune response and measured by the titer ofspecific antibodies detected in the serum.

TABLE 17 ELISA detection of specific IgG titers from murine afterimmunization with PIKA and/or HSV2 gD vaccines Groups of Mice Group 1HSV2gD Group 2 Group 3 3 ug + HSV2gp PIKA Group 4 PIKA 100 ug 3 ug 100ug PBS Optical density absorbance 2.116 0.554 0.085 0.087 405 nm at2,700x dilution

The conclusion drawn is that the addition of the PIKA adjuvant enhancesthe overall immune response to HSV antigen, in particular the specificimmune response, more particularly the adaptive immunity and morespecifically the cell mediated immune response.

Example 1.6 Inactivated H5N1 Whole Virus (Avian Influenza) Antigen

The results in table 18 below are the results of the ELISPOT testdetecting the presence of INF-γ, IL-2 and IL-4 using an inactivated nonpurified H5N1 antigen. The data in table 18 (see also FIGS. 24, 25 and26) represent the ELISPOT reading, the number of spot forming cells,that is, a direct measure of cytokine production.

The distinct increase in the number of spot forming cells with theaddition of the PIKA adjuvant (as compared with the antigen alone)demonstrates that the addition of the PIKA adjuvant to the H5N1 antigenenhances the expression of cytokines INF-γ, IL-2 and IL-4 by culturedspleen cells. The observed expression of cytokines indicates an enhancedadaptive immune response of both a humoral and cell mediated immunityinduced by the presence of the PIKA adjuvant.

TABLE 18 ELISPOT detection of murine splenocytes producing cytokinesafter immunization with vaccines comprising PIKA and/or inactivated H5N1antigen Groups of mice Group 1 H5N1 4 ul + Group 2 Group 3 Group 4 PIKA100 ug H5N1 4 ul PIKA 100 ug PBS No. of cells producing interferon-γ/Stimulated with H5N1 394 372 23 98  2.5 × 10⁵ splenocytes Not stimulated3 1 — 1 No. of cells producing IL-2/ Stimulated with H5N1 135 97  1 22.5 × 10⁵ splenocytes Not stimulated 2 2 — — No. of cells producingIL-4/ Stimulated with H5N1 184 137 10 9 2.5 × 10⁵ splenocytes Notstimulated 9 11 11 17  Unit: Splenocyte spot forming cells

The results of the FACS analysis are presented in table 19 below (seealso FIG. 27). The presence of CD4+ T cells expressing INF-γ in only theformulations containing both PIKA and H5N1 antigen confirms theobservation that the adaptive immune response has reach a stage ofmaturity and that PIKA was instrumental in this process.

TABLE 19 FACS analysis of murine splenocytes after immunization withvaccines comprising PIKA and/or inactivated H5N1 antigen Groups of MiceGroup 1 Group 2 Group 3 H5N1 4 ul + H5N1 PIKA Group 4 PIKA 100 ug 4 ul100 ug NS % CD4+ve cells producing 1.147% 0.263% 0.005% 0.011%interferon-γ

Results of the ELISA test on the blood sample taken prior to sacrifice(Table 20 below and FIG. 28) demonstrate that the presence of PIKAsignificantly enhances the immune response and measured by the titer ofspecific antibodies detected in the serum.

TABLE 20 ELISA detection of specific IgG titers from murine serum afterimmunization with vaccines comprising PIKA and/or inactivated H5N1antigen Groups of Mice Group 1 Group 2 Group 3 H5N1 4 ul + H5N1 PIKAGroup 4 PIKA 100 ug 4 ul 100 ug PBS Optical density absorbance 1.0330.656 0.09 0.079 405 nm at 900x dilution

The conclusion drawn is that the addition of the PIKA adjuvant enhancesthe overall immune response to H5N1 antigen, in particular the specificimmune response, more particularly the adaptive immunity and morespecifically the cell mediated immune response.

Example 2 Inactivated Whole Virus SARS Antigen

The objective of this experiment is to demonstrate that the addition ofPIKA to a SARS antigen enhances the immune response and stimulates thehost's immune system to produce protective SARS specific antibodies.

In this program of research six groups each comprising 4 Balb/c micewere inoculated (peritoneal injection) with a combination of SARSantigen, the antigen plus PIKA (a heterogeneous composition of PIKAmolecules predominantly within a weight range of 66 kDa to 1,200,000kDa), PIKA alone or a control, see table 21 below (see also FIG. 29).

Each group was administered an identical doses on day 0, day 14 and day28. On week six a blood sample was extracted and the serum tested forthe presence of IgG, being a measure of the presence of disease specificantibodies. The blood serum was diluted by a factor of 16,000 times thenthe presence of IgG was measured using an ELISA reader the procedurebeing familiar to those skilled in the art. The output being an opticaldensity (O.D.) reading where the greater the value the greater thepresence of IgG.

The average result for each group, presented in table 21, demonstrates acorrelation between the presence of the PIKA adjuvant and an increase inthe expression of IgG.

TABLE 21 ELISA detection of specific IgG titers from murine serum afterimmunization with vaccines comprising PIKA and/or whole inactivated SARSantigen Groups of mice Group 2 Group 3 Group 1 SARS 10 ug + SARS 10 ug +Group 4 Group 5 SARS 10 ug PIKA 50 ug PIKA 250 ug PIKA 100 ug PBSOptical density 0.26 0.41 0.68 0.09 0.09 absorbtion 405 nm 16,000xdilution

The conclusion is that the presence of PIKA with the SARS antigenincreases the expression of IgG in a dose dependent manner therebyenhancing the immune response of the host.

Example 3 PIKA Vaccine Provides Immune Protection Against H5N1 Infection

The objective of this experiment is to demonstrate that an avianinfluenza vaccine comprising the PIKA adjuvant is able to protectchickens against live avian flu virus infection.

The research was conducted on two groups of 24 SPF chickens each. At tendays old the birds were inoculated subcutaneously in the neck with a 700ul dose of vaccine comprising PIKA (a heterogeneous composition of PIKAmolecules predominantly within a weight range of 66 kDa to 660 kDa) andtwo strains of avian influenza (H5N1 and H9N2). The composition includedantigen and PIKA adjuvant at a ratio of approximately 2:1 antigen to thePIKA adjuvant.

Blood samples were taken from under the wing at 7, 14 and 21 days. Theblood serum from each chicken was tested for the presence of specific H5and H9 antibodies.

At 21 days the birds were challenged with the H5N1 live virus and thenobserved for an additional 14 days. The survival rate of the chickensafter the 14 days exposure to the live H5N1 virus was recorded.

The average result for each group (Table 22 see also FIGS. 30 and 31)demonstrates that the presence of PIKA induces the production ofspecific antigen antibodies.

TABLE 22 ELISA detection of specific antibody titers from chicken serumafter immunization with vaccines comprising PIKA and/or inactivated H5N1antigen Antibody Day 0 Day 7 Day 14 Day 21 H5 0 1.2 1.44 2.4 H9 0 1.72.3 3.9 Units: Optical density reading from ELISA analysis

Of the 24 chickens that were vaccinated with the antigen/PIKAcomposition 21 (83%) survived for 14 days after exposure to the liveH5N1 virus. In the control group of 24 chickens that received no vaccinebut were also exposed to the live H5N1 virus only 4 (17%) were aliveafter 14 days.

The conclusion drawn is that the PIKA vaccine confers a significantlevel of immune protection against the H5N1 virus.

Example 4 PIKA Vaccine Provides Immune Protection Against RabiesInfection

The objective of this research is to demonstrate that a rabies vaccinecomprising the PIKA adjuvant is able to confer protection against arabies infection.

Four groups (designated i, ii, iii and iv) of 20 Balb/c SPF Kunming micewere each challenged with 100 ul of wild rabies virus strain CQ92. Eachgroup receive inoculations of different types of vaccine; i) acomposition of PIKA (a heterogeneous composition of PIKA moleculespredominantly within a weight range of 66 kDa to 660 kDa) andinactivated purified hamster kidney cell rabies antigen in a ratio of1:4 by volume, ii) Sanofi-Aventis' Veroab vero cell inactivated rabies,vaccine iii) the inactivated purified hamster kidney cell rabies vaccinewith an alum adjuvant and iv) control phosphate buffer solution. A 60 uldose of vaccine was administered 30 to 40 minutes, 3 days, 6 days and 9days after infection by subcutaneous injection into the thigh.

The survival rate of each groups presented in table 23. (see also FIG.32).

TABLE 23 Survival rates of mice exposed to wild rabies virus andsubsequent treatment with rabies vaccines Survival Vaccine Mice Number %IPHK plus PIKA 20 16 80% Verorab 20 4 20% IPHK plus alum 20 3 15%Control (PBS) 20 2 10%

The conclusion drawn is that the presence of PIKA significantly enhancesthe immune protection provided by the inactivated purified hamsterkidney cell rabies antigen.

Example 5 PIKA Hepatitis B Vaccine Induces the Production of SpecificAntibodies in Serum

The protocol for the experiment involved the vaccination by subcutaneousinjection of three groups of Balb/c mice (three mice per group) withcompositions of, group A, 4 ug of the hepatitis B surface antigen adwalone, group B, 4 ug of the antigen with 75 ug of the PIKA adjuvant (aheterogeneous composition of PIKA molecules predominantly within aweight range distribution of about 66 kDa to 1,200 kDa) and group C, 100ug of PIKA alone.

The mice were then given an identical booster vaccine by subcutaneousinjection ten to fourteen days after the initial injection. Ten tofourteen days after the booster injection a blood sample was taken andtested for the specific antibody titer using a standard ELISA test knownto those skilled in the art.

The average results for each group presented in table 24 below (and FIG.33) demonstrate that the presence of PIKA enhances the immune responseto the hepatitis B antigen as measured by the titer of specificantibodies observed in the serum sample.

TABLE 24 ELISA detection of specific antibody titers from murine serumafter immunization with vaccines comprising PIKA and/or HBsAg adw Groupsof mice Group 2 Group 3 Group 4 Group 5 Group 1 HBsAg 1 ug + HBsAg 2ug + HBsAg 3 ug + HBsAg 6 ug + Group 6 Group 7 HBsAg 3 ug PIKA 80 ugPIKA 80 ug PIKA 80 ug PIKA 80 ug PIKA 80 ug PBS Optical densityabsorbtion 0.4160 1.9043 2.5040 2.8470 3.0240 0.0680 0.0747 405 nm at12,800x dilution

The conclusion drawn from this example is that an immunogenic substancecomprising PIKA and a hepatitis B antigen induces the production of asignificant immune response as measured by the titer of specificantibodies in the blood serum.

Example 6 PIKA Influenza Vaccine Induces the Production of SpecificAntibodies in Serum

The protocol for the experiment involved the vaccination by subcutaneousinjection of two groups of Balb/c mice (three mice per group) withcompositions of, group A, 4 ug of the Sanofi VAXIGRIP influenza vaccinealone, and group B, 4 ug of the antigen with 100 ug of the PIKA adjuvant(a heterogeneous composition of PIKA molecules predominantly within aweight range distribution of about 66 kDa to 1,200 kDa).

The mice were then given an identical booster vaccine by subcutaneousinjection twenty days after the initial injection. Day 35 after theinitial vaccination a blood sample was taken and tested for the specificantibody titer using a standard ELISA test known to those skilled in theart.

The average results for each group presented in table 25 below (and FIG.34) demonstrate that the presence of PIKA enhances the immune responseto the influenza vaccine antigens as measured by the titer of specificantibodies observed in the serum sample.

TABLE 25 ELISA detection of specific antibody titers from murine serumafter immunization with vaccines comprising PIKA and/or inactivatedsplit influenza antigen Groups of Mice Group 2 Group 1 Flu 4 ug + Group3 Flu 4 ug PIKA 100 ug PIKA 100 ug Optical density absorbtion 1.8392.804 0.087 405 nm at 10x dilution

The conclusion drawn from this example is that an immunogenic substancecomprising PIKA and an influenza antigen induces the production of asignificant immune response as measured by the titer of specificantibodies in the blood serum.

Example 7 PIKA Hepatitis B (Surface Antigen Type adw) Vaccine Induces aTherapeutic Immune Response

The protocol for the experiment involves the inoculation of groups of 4Balb/c mice 6 to 10 weeks old with compositions of a commerciallyavailable HBsAg type adw with and without the PIKA adjuvant (aheterogeneous composition of PIKA molecules predominantly within aweight range distribution of about 66 kDa to 1,200 kDa), PIKA alone, acontrol comprising phosphate buffer solution (PBS).

The mice were administered a prime subcutaneous injection in both sidesof the back 100 ul each side. Actual dosage quantities are provided inthe tables of results below. The mice were then given an identicalbooster vaccine twenty one days after the initial injection. On dayforty two a blood sample was taken, the mice were then sacrificed andtissue samples taken from the spleen for testing.

ELISPOT assays were conducted to enumerate the antigen-specificinterferon-γ secreting T cells. A sample of splenocytes from each mousewas stimulated ex-vivo with either a CD8 T cell peptide epitope fromHBsAg (IPQSLDSWWTSL) at a concentration of 5 ug/ml to measure thepresence of IPQSLDSWWTSL-specific CD8+ cells.

A second sample of splenocytes were restimulated ex-vivo for six dayswith 2 ug/ml with the HBsAg peptide IPQSLDSWWTSL. A ELISPOT assay, using5 ug/ml HBsAg peptide IPQSLDSWWTSL as an ex-vivo stimulant was conductedto detect interferon-γ. This assay was conducted to identify to evaluatethe central memory cell response following immunization.

An ELISA assay was used to measure the presence of HBV antigen specificantibodies in the serum, specifically IgG1 and IgG2a antibodies. NuncImmunoplate Maxisorp plates were coated overnight at 4 deg C. with HBsAg(6 ug/ml in PBS/0.01% Tween 20). The plates were washed with PBS/Tweenand blocked for 2 hours with 5% FCS in PBS. After washing serumdilutions in PBS/Tween were added for 2 hours. After washing the eitherbiotin conjugated rat anti-mouse IgG1 monoclonal antibody 1/3000dilution or the biotin conjugated rat anti-mouse IgG2a monoclonalantibody 1/1500 dilution was added. After washing, streptavidin HRP wasadded (1/10,000 dilution in PBS/Tween) for 1 hour. After washing ABTSsubstrate was added with hydrogen peroxide (1000:1) for 20 minutes. Theoptical density (OD) was then measured at 405 nm the results presentedare the average for each group.

The IgG1 response for mice immunized with HBsAg formulated with the PIKAadjuvant were approximately 5 fold higher than the response for miceimmunized with HBsAg alone. The titer of IgG1 increased in a dosedependent manner (Table 26 FIG. 35).

TABLE 26 ELISA detection of specific IgG1 titers from murine serum afterimmunization with vaccines comprising PIKA and/or HBsAg Group 1 Group 2Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 HBsAg PIKA 50x 150x 450x1,350x 4,050x 12,150x 36,450x 109,350x 3 ug  50 ug 3.285 3.279 3.3243.307 3.307 3.083 2.434 1.383 3 ug 100 ug 3.387 3.416 3.448 3.486 3.5593.368 2.924 1.917 3 ug 200 ug 3.358 3.365 3.424 3.474 3.563 3.351 3.0082.170 3 ug — 3.065 3.065 3.135 2.966 2.814 2.362 1.546 0.790 — 100 ug0.493 0.248 0.157 0.113 0.109 0.120 0.102 0.101 PBS 100 ul 0.136 0.1010.086 0.075 0.080 0.077 0.082 0.087 Unit: Average optical density value

The IgG2a response for mice immunized with HBsAg formulated with thePIKA adjuvant were significantly greater than the response for miceimmunized with HBsAg alone. The titer of IgG2a increased in a dosedependent manner, indicative of an increased Th1 biased immune response(Table 27 FIG. 36).

TABLE 27 ELISA detection of specific IgG1 titers from murine serum afterimmunization with vaccines comprising PIKA and/or HBsAg Group 1 Group 2Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 HBsAg PIKA 50x 150x 450x1,350x 4,050x 12,150x 36,450x 109,350x 3 ug  50 ug 3.159 2.950 2.8602.061 1.234 0.582 0.265 0.135 3 ug 100 ug 3.411 3.170 3.137 2.101 1.1270.488 0.221 0.122 3 ug 200 ug 3.397 3.346 3.560 2.636 1.955 1.005 0.4550.203 3 ug — 0.122 0.098 0.087 0.072 0.069 0.065 0.069 0.064 — 100 ug0.108 0.086 0.078 0.070 0.067 0.068 0.067 0.065 PBS 100 ul 0.088 0.0780.074 0.067 0.066 0.066 0.066 0.067 Unit: Average optical density value

The ELISPOT assay of the CD8 peptide epitope specific ex-vivostimulation showed an undetectable response for the mice immunized withHBsAg alone. By contrast, cells expressing interferon-γ were readilydetectable after immunization with HBsAg formulated with PIKA in a dosedependent manner indicating that PIKA enhances a therapeutic immuneresponse (Table 28 FIG. 37).

TABLE 28 ELISPOT detection of murine splenocytes producing interferon-γafter immunization with vaccines comprising PIKA and/or HBsAg Group 1Group 2 Group 3 Group 4 Group 5 HBsAg 3 ug 3 ug 3 ug 3 ug — PIKA — 50 ug100 ug 200 ug 100 ug Stimulation with IPQ No long term 209 229 490 53521 peptide 5 ug/ml stimulation Stimulation with IPQ Restimulate for 6 282 89 48 3 peptide 5 ug/ml days with 2 ug/ml IPQ peptide Unit:Splenocyte spot forming cells

Further the ELISPOT assay conducted after the cultivation of thesplenocytes for six days demonstrated that the number of splenocytesproducing interferon-γ from the mice vaccinated with formulationscomprising HBsAg and the PIKA adjuvant were twice the number ofsplenocytes from mice administered the HBsAg alone. The results confirmthat the presence of PIKA enhances the activation of central memorycells (Table 28 FIG. 38).

1. An immunogenic composition comprising: (a) a polynucleotide adjuvantcomprising: a polyriboinosinic-polyribocytidylic acid (PIC), at leastone an antibiotic, and at least one positive ion; and (b) at least oneantigen; wherein the composition is formulated for sustained release. 2.An immunogenic composition comprising: (a) a polynucleotide adjuvantcomprising: a polyriboinosinic-polyribocytidylic acid (PIC), at leastone an antibiotic, and at least one positive ion; and (b) at least oneviral antigen; wherein the antigen is an antigen of adeniviridae,arenaviridae, astroviridae, bunyaviridae, cliciviridae, flaviviridae,hepatitis delta virus, hepeviridae, mononegavirales, nidovirales,piconaviridae, orthomyxoviridae, papillomaviridae, parvoviridae,polyomaviridae, poxyiridae, reoviridae, retroviridae or togaviridae. 3.An immunogenic composition comprising: (a) a polynucleotide adjuvantcomprising: a polyriboinosinic-polyribocytidylic acid (PIC), at leastone an antibiotic, and at least one positive ion; and (b) at least onebacterial antigen wherein the antigen is an antigen of actinobacteria,chlamydiae, filmicutes, proteobacteria or spirochaetes.
 4. Animmunogenic composition comprising: (a) a polynucleotide adjuvantcomprising: a polyriboinosinic-polyribocytidylic acid (PIC), at leastone an antibiotic, and at least one positive ion; and (b) at least onefungal antigen wherein the antigen is an antigen of ascomycota orbasidiomycota.
 5. An immunogenic composition comprising: (a) apolynucleotide adjuvant comprising: a polyriboinosinic-polyribocytidylicacid (PIC), at least one an antibiotic, and at least one positive ion;and (b) at least one parasitic antigen wherein the antigen is an antigenof phylum sarcomastigophora, phylum apiicomplexa, phylum ciliophora,phylum plathyhelminthes, phylum nematoda or phylum arthropoda.
 6. Animmunogenic composition comprising: (a) a polynucleotide adjuvantcomprising: a polyriboinosinic-polyribocytidylic acid (PIC), at leastone an antibiotic, and at least one positive ion; and (b) at least onecancer antigen wherein the antigen is a cancer antigen of bone, brain,breast, digestive/gastrointestinal, endocrine, eye, genitourinary, germcell, gynecologic, head and neck, hematological/blood, lung,musculoskeletal, neurologic, respiratory/thoracic or skin cancer.
 7. Animmunogenic composition comprising: (a) a polynucleotide adjuvantcomprising: a polyriboinosinic-polyribocytidylic acid (PIC), at leastone an antibiotic, and at least one positive ion; and (b) a combinationof two or more antigens wherein the antigens are antigens ofadeniviridae, arenaviridae, astroviridae, bunyaviridae, cliciviridae,flaviviridae, hepatitis delta virus, hepeviridae, mononegavirales,nidovirales, piconaviridae, orthomyxoviridae, papillomaviridae,parvoviridae, polyomaviridae, poxyiridae, reoviridae, retroviridae,togaviridae, actinobacteria, chlamydiae, firmicutes, proteobacteria orspirochaetes, ascomycotaor basidiomycota, phylum sarcomastigophora,phylum apiicomplexa, phylum ciliophora, phylum plathyhelminthes, phylumnematoda and/or phylum arthropoda.
 8. The immunogenic compositionaccording to any one of claims 1 to 7, wherein the composition comprisespolynucleotide adjuvant composition molecules heterogeneous formolecular weight, wherein the molecular weight is at least 66,000Daltons.
 9. The immunogenic composition according to any one of claims 1to 8, wherein the composition comprises polynucleotide adjuvantcomposition molecules heterogeneous for molecular weight, wherein themolecular weight is from about 66,000 to 1,200,000 Daltons.
 10. Theimmunogenic composition according to any one of claims 1 to 9, whereinthe composition comprises polynucleotide adjuvant composition moleculesheterogeneous for molecular weight, wherein the molecular weight is atleast 150,000 Daltons.
 11. The immunogenic composition of any one ofclaims 1 to 10, further comprising at least one immunomodulator.
 12. Theimmunogenic composition of any one of claims 1 to 11, wherein theantigen is an inactivated microorganism, attenuated microorganism,recombinant polypeptide and/or synthetic polypeptide.
 13. Theimmunogenic composition of any one of claims 1 to 12 wherein theimmunogenic composition or the PIKA adjuvant contained in theimmunogenic composition, is in the form of a liquid, liquid solution,liquid drops, a solid, capsules, emulsions, suspensions, elixirs,creams, suppositories, gels, soft capsules, sprays, inhalants, aerosols,tablets, coated tablets, microcapsules, suppositories, dragees, syrups,slurries, enemas, granules, powders, tablets or lozenges.
 14. Theimmunogenic composition of any one of claims 1 to 12 wherein at leastone of the adjuvant composition or the immunogenic composition isfreeze-dried.
 15. A kit comprising the immunogenic composition of anyone of claims 1 to
 14. 16. A method for enhancing an immune response ina host to an antigen, the method comprising administering to the hostthe immunogenic composition of any of claims 1 to
 14. 17. The method ofclaim 16, wherein the host has an infectious disease and saidadministering of the antigenic compound elicits an immune responseagainst the pathogen causing the infectious disease.
 18. The method ofclaim 16 or claim 17, wherein said administering is by parenteralinjection, intramuscular injection, intraperitoneal injection,intravenous injection, subcutaneous injection, topical delivery,transdermal delivery or intradermal delivery.
 19. The use of theimmunogenic composition of any of claim 1 to 14 for the preparation of amedicament for enhancing the immune response of a host to the antigen.20. The use of claim 19, wherein the host has an infectious disease andthe administering of the antigen to the host elicits an immune responseagainst the pathogen causing the infectious disease.
 21. The use ofclaim 20, wherein the administering is by parenteral injection,intramuscular injection, intraperitoneal injection, intravenousinjection, subcutaneous injection, topical delivery, transdermaldelivery or intradermal delivery.
 22. The method of any one of claims 16to 18 and the use of any one of claims 19 to 21, wherein the host ishuman.
 23. The method of any one of claims 16 to 18 and the use of anyone of claims 19 to 21, wherein the host is a non-human animal.